<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2025 Workiva-->
<!--r:19e77f6d-4fb4-4346-a843-0b1cefbf3434,g:4ccf593f-e13a-4058-9e00-bc5e55f7a196-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.autolus.com/role/CoverPage" xlink:type="simple" xlink:href="autl-20250630.xsd#CoverPage"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/CoverPage" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentInformationTable_022613f6-51f9-43e1-9f5d-8a9724489dc8" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentInformationTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentInformationTable_022613f6-51f9-43e1-9f5d-8a9724489dc8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_dei_DocumentInformationTable_022613f6-51f9-43e1-9f5d-8a9724489dc8" xlink:to="loc_us-gaap_StatementClassOfStockAxis_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f" xlink:to="loc_us-gaap_ClassOfStockDomain_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_c29638ae-df15-44f1-aa63-8bead7e66b54" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_ded9477c-80e4-4c41-8fb6-9bcb9e4d690f" xlink:to="loc_us-gaap_ClassOfStockDomain_c29638ae-df15-44f1-aa63-8bead7e66b54" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_AdrMember_c7af8f3c-3fa3-42af-9791-a2aabe8bd5e7" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AdrMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_c29638ae-df15-44f1-aa63-8bead7e66b54" xlink:to="loc_dei_AdrMember_c7af8f3c-3fa3-42af-9791-a2aabe8bd5e7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_OrdinarySharesMember_948c6846-5fd6-482b-94d3-24b16f4800d3" xlink:href="autl-20250630.xsd#autl_OrdinarySharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_c29638ae-df15-44f1-aa63-8bead7e66b54" xlink:to="loc_autl_OrdinarySharesMember_948c6846-5fd6-482b-94d3-24b16f4800d3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentType_1f1aff8b-a2e3-4fb7-a6b3-bc7a3f525755" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentType"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentType_1f1aff8b-a2e3-4fb7-a6b3-bc7a3f525755" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentQuarterlyReport_bb9cd21b-0fb7-410f-a7ca-a64c8901065a" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentQuarterlyReport"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentQuarterlyReport_bb9cd21b-0fb7-410f-a7ca-a64c8901065a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentPeriodEndDate_7c89921e-d517-4975-bb5c-fc964b211e54" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentPeriodEndDate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentPeriodEndDate_7c89921e-d517-4975-bb5c-fc964b211e54" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentTransitionReport_3ab72f5e-2a59-468d-b918-b73430bfc00e" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentTransitionReport"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentTransitionReport_3ab72f5e-2a59-468d-b918-b73430bfc00e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityFileNumber_b61bf937-23ba-448a-bd0f-752fcfa4df65" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFileNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityFileNumber_b61bf937-23ba-448a-bd0f-752fcfa4df65" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityRegistrantName_3511003f-06e6-4fb8-82f2-35091d9c9b12" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityRegistrantName"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityRegistrantName_3511003f-06e6-4fb8-82f2-35091d9c9b12" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityIncorporationStateCountryCode_ebe64972-52ed-41a7-8dec-6940a185ffa4" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityIncorporationStateCountryCode"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityIncorporationStateCountryCode_ebe64972-52ed-41a7-8dec-6940a185ffa4" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressAddressLine1_c344865b-824a-4ea3-9a93-f34fb1eaface" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressAddressLine1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityAddressAddressLine1_c344865b-824a-4ea3-9a93-f34fb1eaface" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressAddressLine2_cc01de5c-8407-46e1-8668-ce2ffa8b7f7f" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressAddressLine2"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityAddressAddressLine2_cc01de5c-8407-46e1-8668-ce2ffa8b7f7f" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressCityOrTown_c52bb13b-bac9-4cd6-a363-2ca180074cb0" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressCityOrTown"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityAddressCityOrTown_c52bb13b-bac9-4cd6-a363-2ca180074cb0" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressPostalZipCode_9baa8599-9091-4653-975d-586e7ae728b6" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressPostalZipCode"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityAddressPostalZipCode_9baa8599-9091-4653-975d-586e7ae728b6" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressCountry_3810c483-4594-4e19-91e5-eed30eb394ae" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressCountry"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityAddressCountry_3810c483-4594-4e19-91e5-eed30eb394ae" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_CityAreaCode_6673187f-6a18-4b1d-83dd-ad89a22233c4" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CityAreaCode"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_CityAreaCode_6673187f-6a18-4b1d-83dd-ad89a22233c4" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_LocalPhoneNumber_b731f2f5-ab15-4cc8-9c3c-16f1bf04351d" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_LocalPhoneNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_LocalPhoneNumber_b731f2f5-ab15-4cc8-9c3c-16f1bf04351d" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_Security12bTitle_e5ec3359-b828-4940-9d81-590f59ca9db6" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_Security12bTitle"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_Security12bTitle_e5ec3359-b828-4940-9d81-590f59ca9db6" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_TradingSymbol_ab8687ad-1a92-40d8-ad39-4fe48af26ec7" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_TradingSymbol"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_TradingSymbol_ab8687ad-1a92-40d8-ad39-4fe48af26ec7" xlink:type="arc" order="15"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_SecurityExchangeName_61217dd4-4139-423b-ac8e-ea9d13c38558" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_SecurityExchangeName"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_SecurityExchangeName_61217dd4-4139-423b-ac8e-ea9d13c38558" xlink:type="arc" order="16"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityCurrentReportingStatus_bcf8cffa-1435-4fd6-bab2-ea141cf3127d" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCurrentReportingStatus"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityCurrentReportingStatus_bcf8cffa-1435-4fd6-bab2-ea141cf3127d" xlink:type="arc" order="17"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityInteractiveDataCurrent_b980ee72-d0c8-46eb-8a1e-24ebf0b7d48a" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityInteractiveDataCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityInteractiveDataCurrent_b980ee72-d0c8-46eb-8a1e-24ebf0b7d48a" xlink:type="arc" order="18"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityFilerCategory_1d392739-6467-4d88-aba0-408fb5bfcce8" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFilerCategory"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityFilerCategory_1d392739-6467-4d88-aba0-408fb5bfcce8" xlink:type="arc" order="19"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntitySmallBusiness_66e2b084-4325-4b4d-83f7-6db978d6305e" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntitySmallBusiness"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntitySmallBusiness_66e2b084-4325-4b4d-83f7-6db978d6305e" xlink:type="arc" order="20"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityEmergingGrowthCompany_1c2a2b2e-21ea-4448-b08e-e21ed7183ea6" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityEmergingGrowthCompany"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityEmergingGrowthCompany_1c2a2b2e-21ea-4448-b08e-e21ed7183ea6" xlink:type="arc" order="21"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityShellCompany_fc8e5925-108e-4d6e-80fe-483a0aa42f4d" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityShellCompany"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityShellCompany_fc8e5925-108e-4d6e-80fe-483a0aa42f4d" xlink:type="arc" order="22"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityCommonStockSharesOutstanding_5d997453-8dfb-4a18-bc5c-fd347e29459f" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityCommonStockSharesOutstanding_5d997453-8dfb-4a18-bc5c-fd347e29459f" xlink:type="arc" order="23"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityCentralIndexKey_1740d995-2894-45cd-92eb-a4f91ddd7f48" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCentralIndexKey"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_EntityCentralIndexKey_1740d995-2894-45cd-92eb-a4f91ddd7f48" xlink:type="arc" order="24"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_AmendmentFlag_3c043f0f-1d3c-44e4-a801-7398c6bf58ca" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AmendmentFlag"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_AmendmentFlag_3c043f0f-1d3c-44e4-a801-7398c6bf58ca" xlink:type="arc" order="25"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentFiscalYearFocus_f5bf3ac9-d1ef-42ea-a654-3c4546166cd9" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalYearFocus"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentFiscalYearFocus_f5bf3ac9-d1ef-42ea-a654-3c4546166cd9" xlink:type="arc" order="26"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentFiscalPeriodFocus_0306325a-3ada-4854-82cf-e118549cf2a3" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalPeriodFocus"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_DocumentFiscalPeriodFocus_0306325a-3ada-4854-82cf-e118549cf2a3" xlink:type="arc" order="27"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_CurrentFiscalYearEndDate_56449751-9a6a-4b19-b428-62b1eca4f6aa" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CurrentFiscalYearEndDate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_CurrentFiscalYearEndDate_56449751-9a6a-4b19-b428-62b1eca4f6aa" xlink:type="arc" order="28"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_NoTradingSymbolFlag_961f9b6b-96e5-48de-9c37-bb228ca7a47b" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_NoTradingSymbolFlag"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_DocumentInformationLineItems_03a58566-8099-48fd-9110-b9b98bc98ffd" xlink:to="loc_dei_NoTradingSymbolFlag_961f9b6b-96e5-48de-9c37-bb228ca7a47b" xlink:type="arc" order="29"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/UnauditedCondensedConsolidatedBalanceSheets" xlink:type="simple" xlink:href="autl-20250630.xsd#UnauditedCondensedConsolidatedBalanceSheets"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/UnauditedCondensedConsolidatedBalanceSheets" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_43bf2f0e-4e58-4b73-9754-1f2383232a21" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_f309ef2f-fb2a-4c76-956e-fbf8b4c81ef9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_43bf2f0e-4e58-4b73-9754-1f2383232a21" xlink:to="loc_us-gaap_StatementTable_f309ef2f-fb2a-4c76-956e-fbf8b4c81ef9" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_8ca35e2e-0967-497e-91c4-ec20e32ded95" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_f309ef2f-fb2a-4c76-956e-fbf8b4c81ef9" xlink:to="loc_us-gaap_StatementClassOfStockAxis_8ca35e2e-0967-497e-91c4-ec20e32ded95" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_8ca35e2e-0967-497e-91c4-ec20e32ded95_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_8ca35e2e-0967-497e-91c4-ec20e32ded95" xlink:to="loc_us-gaap_ClassOfStockDomain_8ca35e2e-0967-497e-91c4-ec20e32ded95_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_8ca35e2e-0967-497e-91c4-ec20e32ded95" xlink:to="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_OrdinarySharesMember_b8cce983-5bd6-46cd-a01b-192b9827bbde" xlink:href="autl-20250630.xsd#autl_OrdinarySharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:to="loc_autl_OrdinarySharesMember_b8cce983-5bd6-46cd-a01b-192b9827bbde" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredSharesMember_1379691b-c341-48a9-8bb7-e1d829f88646" xlink:href="autl-20250630.xsd#autl_DeferredSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:to="loc_autl_DeferredSharesMember_1379691b-c341-48a9-8bb7-e1d829f88646" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassBSharesMember_de2584df-8e9a-41e1-9f5c-8a64ac9449a3" xlink:href="autl-20250630.xsd#autl_DeferredClassBSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:to="loc_autl_DeferredClassBSharesMember_de2584df-8e9a-41e1-9f5c-8a64ac9449a3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassCSharesMember_dcec97d1-2915-4032-8afa-d3de286856da" xlink:href="autl-20250630.xsd#autl_DeferredClassCSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_9e853356-9b30-4f54-892b-ed4e4c296750" xlink:to="loc_autl_DeferredClassCSharesMember_dcec97d1-2915-4032-8afa-d3de286856da" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsAbstract_634cc580-e2f3-4c05-9ac6-18c2eaa597f2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_43bf2f0e-4e58-4b73-9754-1f2383232a21" xlink:to="loc_us-gaap_AssetsAbstract_634cc580-e2f3-4c05-9ac6-18c2eaa597f2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsCurrentAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsAbstract_634cc580-e2f3-4c05-9ac6-18c2eaa597f2" xlink:to="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsAtCarryingValue_8f3aceff-9495-4a2a-a8fd-417f3d710354" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_CashAndCashEquivalentsAtCarryingValue_8f3aceff-9495-4a2a-a8fd-417f3d710354" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecuritiesCurrent_9234428d-4e68-48de-9687-d734b7f06b20" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecuritiesCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecuritiesCurrent_9234428d-4e68-48de-9687-d734b7f06b20" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashCurrent_ffd939c6-93f0-4b76-8359-f1631f8b8326" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_RestrictedCashCurrent_ffd939c6-93f0-4b76-8359-f1631f8b8326" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsAndOtherReceivablesNetCurrent_14cd4599-ea63-47d6-a172-9a9ea809f6b5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccountsAndOtherReceivablesNetCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_AccountsAndOtherReceivablesNetCurrent_14cd4599-ea63-47d6-a172-9a9ea809f6b5" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InventoryNet_a76f3abd-eb2a-4278-9466-f10601ae59b5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InventoryNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_InventoryNet_a76f3abd-eb2a-4278-9466-f10601ae59b5" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PrepaidExpenseAndOtherAssetsCurrent_c1908d2a-fa3b-472d-b63d-2fbc39b9574a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_PrepaidExpenseAndOtherAssetsCurrent_c1908d2a-fa3b-472d-b63d-2fbc39b9574a" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsCurrent_e4f943aa-70db-46a9-b13a-27f9ac7a4b46" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsCurrentAbstract_f9a4c098-55ce-4ae4-b949-5ac4525ed5f5" xlink:to="loc_us-gaap_AssetsCurrent_e4f943aa-70db-46a9-b13a-27f9ac7a4b46" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsNoncurrentAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsAbstract_634cc580-e2f3-4c05-9ac6-18c2eaa597f2" xlink:to="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentNet_6e5270b4-fdb1-4d63-bcd2-a3654861144c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentNet_6e5270b4-fdb1-4d63-bcd2-a3654861144c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsNet_c1d6f9d5-b78c-4f8c-8353-45b386345d61" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsNet_c1d6f9d5-b78c-4f8c-8353-45b386345d61" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PrepaidExpenseAndOtherAssetsNoncurrent_7b6f686b-f491-459d-be8f-47308ce38278" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrepaidExpenseAndOtherAssetsNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_PrepaidExpenseAndOtherAssetsNoncurrent_7b6f686b-f491-459d-be8f-47308ce38278" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseRightOfUseAsset_1833a481-ddc4-4858-85f0-602a34d03a7a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseRightOfUseAsset"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_OperatingLeaseRightOfUseAsset_1833a481-ddc4-4858-85f0-602a34d03a7a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DepositsAssetsNoncurrent_8d0e3333-8939-4120-825a-7dd98010401c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DepositsAssetsNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_DepositsAssetsNoncurrent_8d0e3333-8939-4120-825a-7dd98010401c" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredIncomeTaxAssetsNet_216163ae-e5c4-438f-ac24-30f7088ec160" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredIncomeTaxAssetsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsNoncurrentAbstract_58da361f-f860-4e5c-91d6-7d0a268d1ae5" xlink:to="loc_us-gaap_DeferredIncomeTaxAssetsNet_216163ae-e5c4-438f-ac24-30f7088ec160" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Assets_6ea11a28-2fd9-449d-a94f-e50ce3f83e17" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_Assets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsAbstract_634cc580-e2f3-4c05-9ac6-18c2eaa597f2" xlink:to="loc_us-gaap_Assets_6ea11a28-2fd9-449d-a94f-e50ce3f83e17" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesAndStockholdersEquityAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_43bf2f0e-4e58-4b73-9754-1f2383232a21" xlink:to="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesCurrentAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsPayableCurrent_da245d4e-0046-4297-902f-aaeb7680e274" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccountsPayableCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_us-gaap_AccountsPayableCurrent_da245d4e-0046-4297-902f-aaeb7680e274" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccruedLiabilitiesCurrent_97ace992-345d-49cb-8f6d-4eaaf634339c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccruedLiabilitiesCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_us-gaap_AccruedLiabilitiesCurrent_97ace992-345d-49cb-8f6d-4eaaf634339c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractWithCustomerLiabilityDeferredRevenueCurrent_f3d788a3-f112-4d05-b5e0-e3eb64af216f" xlink:href="autl-20250630.xsd#autl_ContractWithCustomerLiabilityDeferredRevenueCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_autl_ContractWithCustomerLiabilityDeferredRevenueCurrent_f3d788a3-f112-4d05-b5e0-e3eb64af216f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseLiabilityCurrent_5d9d35f9-e89a-4706-9767-347877865b93" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilityCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_us-gaap_OperatingLeaseLiabilityCurrent_5d9d35f9-e89a-4706-9767-347877865b93" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityCurrent_82751160-7af1-4916-b0e6-ad0b1c194a7e" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityCurrent_82751160-7af1-4916-b0e6-ad0b1c194a7e" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesCurrent_ec414383-9100-4d2a-a98f-4310afe5109a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesCurrentAbstract_a78a8917-68f1-4a57-9e4e-8703af824a01" xlink:to="loc_us-gaap_LiabilitiesCurrent_ec414383-9100-4d2a-a98f-4310afe5109a" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesNoncurrentAbstract_f95667cb-e576-4e6f-b2d3-27ef54ea5d62" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesNoncurrentAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_LiabilitiesNoncurrentAbstract_f95667cb-e576-4e6f-b2d3-27ef54ea5d62" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseLiabilityNoncurrent_38323e70-3c83-4016-ab73-dbcc9d112b93" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesNoncurrentAbstract_f95667cb-e576-4e6f-b2d3-27ef54ea5d62" xlink:to="loc_us-gaap_OperatingLeaseLiabilityNoncurrent_38323e70-3c83-4016-ab73-dbcc9d112b93" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityNoncurrent_ccd1fc40-e353-44d4-bd5f-1ec9fcc25d9d" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesNoncurrentAbstract_f95667cb-e576-4e6f-b2d3-27ef54ea5d62" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityNoncurrent_ccd1fc40-e353-44d4-bd5f-1ec9fcc25d9d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherLiabilitiesNoncurrent_3c7aaf3d-cae9-4b31-a2e2-e5a4e18c80c5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherLiabilitiesNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesNoncurrentAbstract_f95667cb-e576-4e6f-b2d3-27ef54ea5d62" xlink:to="loc_us-gaap_OtherLiabilitiesNoncurrent_3c7aaf3d-cae9-4b31-a2e2-e5a4e18c80c5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Liabilities_881b54b8-e477-4366-b063-991d7292839d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_Liabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_Liabilities_881b54b8-e477-4366-b063-991d7292839d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommitmentsAndContingencies_de279a8d-d620-45a2-8a47-aae0f5a9de54" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommitmentsAndContingencies"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_CommitmentsAndContingencies_de279a8d-d620-45a2-8a47-aae0f5a9de54" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquityAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockValue_d02858b4-a7d6-49eb-aef8-0cf18ad341b8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:to="loc_us-gaap_CommonStockValue_d02858b4-a7d6-49eb-aef8-0cf18ad341b8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapital_80b8b312-da7b-4e25-ae33-a846085d0303" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapital"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:to="loc_us-gaap_AdditionalPaidInCapital_80b8b312-da7b-4e25-ae33-a846085d0303" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax_ffc5ab26-cc76-4ca0-9bb4-e671ad0a9fe0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossNetOfTax_ffc5ab26-cc76-4ca0-9bb4-e671ad0a9fe0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsAccumulatedDeficit_d25aac48-e1ff-46eb-bc5f-924bb1a6f30d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsAccumulatedDeficit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:to="loc_us-gaap_RetainedEarningsAccumulatedDeficit_d25aac48-e1ff-46eb-bc5f-924bb1a6f30d" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_6a3872e5-d843-417b-8b29-b3438d4590c2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_119f0ff8-6829-42ce-b62c-876af4746c3e" xlink:to="loc_us-gaap_StockholdersEquity_6a3872e5-d843-417b-8b29-b3438d4590c2" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesAndStockholdersEquity_887755bc-3862-43a5-81df-0685e1cc922c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesAndStockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesAndStockholdersEquityAbstract_11f6439a-4ab5-41e2-9611-a0c7a06017ea" xlink:to="loc_us-gaap_LiabilitiesAndStockholdersEquity_887755bc-3862-43a5-81df-0685e1cc922c" xlink:type="arc" order="6"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/UnauditedCondensedConsolidatedBalanceSheetsParenthetical" xlink:type="simple" xlink:href="autl-20250630.xsd#UnauditedCondensedConsolidatedBalanceSheetsParenthetical"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/UnauditedCondensedConsolidatedBalanceSheetsParenthetical" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_799ee9f3-48cb-4b60-a47f-d7a4a5420ab0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_21b98ec0-778b-4a43-9c9b-1303bd5d4ddd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_799ee9f3-48cb-4b60-a47f-d7a4a5420ab0" xlink:to="loc_us-gaap_StatementTable_21b98ec0-778b-4a43-9c9b-1303bd5d4ddd" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_21b98ec0-778b-4a43-9c9b-1303bd5d4ddd" xlink:to="loc_us-gaap_StatementClassOfStockAxis_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4" xlink:to="loc_us-gaap_ClassOfStockDomain_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_8647202b-5c0e-4f65-9fc1-7e4eeca3d8f4" xlink:to="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_OrdinarySharesMember_fe4d576c-1001-4297-8ec7-e87e81b19d3e" xlink:href="autl-20250630.xsd#autl_OrdinarySharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:to="loc_autl_OrdinarySharesMember_fe4d576c-1001-4297-8ec7-e87e81b19d3e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredSharesMember_3bd92a38-de78-42cd-a43a-7bf300cc26a1" xlink:href="autl-20250630.xsd#autl_DeferredSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:to="loc_autl_DeferredSharesMember_3bd92a38-de78-42cd-a43a-7bf300cc26a1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassBSharesMember_3106d5c9-3b1b-4e4b-8f29-487ed33ce086" xlink:href="autl-20250630.xsd#autl_DeferredClassBSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:to="loc_autl_DeferredClassBSharesMember_3106d5c9-3b1b-4e4b-8f29-487ed33ce086" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassCSharesMember_647f5404-aaea-4b35-bfa8-7796eb2b8e0b" xlink:href="autl-20250630.xsd#autl_DeferredClassCSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_641ae523-fdde-4150-86fb-a51a4d5ff9cb" xlink:to="loc_autl_DeferredClassCSharesMember_647f5404-aaea-4b35-bfa8-7796eb2b8e0b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquityAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_799ee9f3-48cb-4b60-a47f-d7a4a5420ab0" xlink:to="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockParOrStatedValuePerShare_b46f452c-a778-4457-8762-89ae5908ea04" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockParOrStatedValuePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:to="loc_us-gaap_CommonStockParOrStatedValuePerShare_b46f452c-a778-4457-8762-89ae5908ea04" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesAuthorized_257fe8ee-23ae-44f8-89f9-3c6982dfc4cf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesAuthorized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:to="loc_us-gaap_CommonStockSharesAuthorized_257fe8ee-23ae-44f8-89f9-3c6982dfc4cf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesIssued_d5617c5a-d76d-449f-bf89-56b8b2711dc9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:to="loc_us-gaap_CommonStockSharesIssued_d5617c5a-d76d-449f-bf89-56b8b2711dc9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_7f89f7a1-f125-4682-82aa-ce9add2b1b0a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StockholdersEquityAbstract_d9760283-d749-41a9-8486-3a7003da79bd" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_7f89f7a1-f125-4682-82aa-ce9add2b1b0a" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/UnauditedCondensedConsolidatedStatementsofOperationsandComprehensiveLoss" xlink:type="simple" xlink:href="autl-20250630.xsd#UnauditedCondensedConsolidatedStatementsofOperationsandComprehensiveLoss"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/UnauditedCondensedConsolidatedStatementsofOperationsandComprehensiveLoss" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_0ee34091-5113-4fde-9bfa-5b4d9f8a6907" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_StatementTable_0ee34091-5113-4fde-9bfa-5b4d9f8a6907" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_63747985-028e-4fd0-b7eb-329f226f4711" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_0ee34091-5113-4fde-9bfa-5b4d9f8a6907" xlink:to="loc_srt_ProductOrServiceAxis_63747985-028e-4fd0-b7eb-329f226f4711" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_63747985-028e-4fd0-b7eb-329f226f4711_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_63747985-028e-4fd0-b7eb-329f226f4711" xlink:to="loc_srt_ProductsAndServicesDomain_63747985-028e-4fd0-b7eb-329f226f4711_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_048a29a6-fb6c-4da0-b364-3b0a997cc607" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_63747985-028e-4fd0-b7eb-329f226f4711" xlink:to="loc_srt_ProductsAndServicesDomain_048a29a6-fb6c-4da0-b364-3b0a997cc607" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProductMember_67aeb0e8-4de5-4287-ab3b-d78baaf38cab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProductMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_048a29a6-fb6c-4da0-b364-3b0a997cc607" xlink:to="loc_us-gaap_ProductMember_67aeb0e8-4de5-4287-ab3b-d78baaf38cab" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicenseMember_745a67b4-fb47-4074-9636-5c3a4877cbc6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LicenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_048a29a6-fb6c-4da0-b364-3b0a997cc607" xlink:to="loc_us-gaap_LicenseMember_745a67b4-fb47-4074-9636-5c3a4877cbc6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_b947cdde-e963-4cb4-a549-64103f2ca897" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_b947cdde-e963-4cb4-a549-64103f2ca897" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpensesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfGoodsAndServicesSold_c4dfcbb5-3ac5-4588-89dc-50274d9ea74a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfGoodsAndServicesSold"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:to="loc_us-gaap_CostOfGoodsAndServicesSold_c4dfcbb5-3ac5-4588-89dc-50274d9ea74a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpense_347a2bcf-3aa2-40ef-af0e-503938e18253" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpense_347a2bcf-3aa2-40ef-af0e-503938e18253" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingGeneralAndAdministrativeExpense_2479f9fe-effe-4895-bbd7-e0fc8837e164" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingGeneralAndAdministrativeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:to="loc_us-gaap_SellingGeneralAndAdministrativeExpense_2479f9fe-effe-4895-bbd7-e0fc8837e164" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GainLossOnDispositionOfAssets_26bc816e-588f-4f26-913f-7f31c982b411" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GainLossOnDispositionOfAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:to="loc_us-gaap_GainLossOnDispositionOfAssets_26bc816e-588f-4f26-913f-7f31c982b411" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseImpairmentLoss_41a56455-cfa7-41c5-8f35-bf4f8ae4d1e3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseImpairmentLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_dd62d27d-f5f7-43bf-bd40-a719ef608979" xlink:to="loc_us-gaap_OperatingLeaseImpairmentLoss_41a56455-cfa7-41c5-8f35-bf4f8ae4d1e3" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_32dcdd9c-c310-4c7d-aaf1-60aea067dfaf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_OperatingIncomeLoss_32dcdd9c-c310-4c7d-aaf1-60aea067dfaf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonoperatingIncomeExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherNonoperatingIncomeExpense_788d7373-58c3-4e00-b13a-72cb7c626503" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherNonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:to="loc_us-gaap_OtherNonoperatingIncomeExpense_788d7373-58c3-4e00-b13a-72cb7c626503" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignCurrencyTransactionGainLossBeforeTax_1050a352-423d-4d0c-ab24-e7940f09dc09" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCurrencyTransactionGainLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:to="loc_us-gaap_ForeignCurrencyTransactionGainLossBeforeTax_1050a352-423d-4d0c-ab24-e7940f09dc09" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentIncomeInterest_2ba49daa-071e-4e44-8e5b-585bf659e706" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InvestmentIncomeInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:to="loc_us-gaap_InvestmentIncomeInterest_2ba49daa-071e-4e44-8e5b-585bf659e706" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestExpenseNonoperating_13e1e662-85f8-4de2-9043-bf8f9e4c7061" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterestExpenseNonoperating"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:to="loc_us-gaap_InterestExpenseNonoperating_13e1e662-85f8-4de2-9043-bf8f9e4c7061" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonoperatingIncomeExpense_e5da14fe-3102-431a-97a9-98310f5ba778" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NonoperatingIncomeExpenseAbstract_9efa9743-3960-41c9-9439-84bc9b4823e9" xlink:to="loc_us-gaap_NonoperatingIncomeExpense_e5da14fe-3102-431a-97a9-98310f5ba778" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_1b38b2e9-891f-46c6-a39f-820a234d2bc2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_1b38b2e9-891f-46c6-a39f-820a234d2bc2" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_294c696e-b79c-459b-87d4-bc00e1a18764" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_294c696e-b79c-459b-87d4-bc00e1a18764" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_f1661ce9-3fde-420c-a40a-7d5908746250" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_NetIncomeLoss_f1661ce9-3fde-420c-a40a-7d5908746250" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTaxAbstract_19834198-99c5-4597-b444-f9f709e54015" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComprehensiveIncomeNetOfTaxAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTaxAbstract_19834198-99c5-4597-b444-f9f709e54015" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_cb0d5623-021c-463c-aa14-b5943b00e019" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxAbstract_19834198-99c5-4597-b444-f9f709e54015" xlink:to="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_cb0d5623-021c-463c-aa14-b5943b00e019" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax_df9e9fc5-c440-4876-9326-811c739eec62" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxAbstract_19834198-99c5-4597-b444-f9f709e54015" xlink:to="loc_us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax_df9e9fc5-c440-4876-9326-811c739eec62" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_8e8eaa46-f8b9-4a8a-920e-c3f9bde1fcd6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxAbstract_19834198-99c5-4597-b444-f9f709e54015" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_8e8eaa46-f8b9-4a8a-920e-c3f9bde1fcd6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTax_8bcf0927-ee20-4bb4-b4d2-3f38ef983dba" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ComprehensiveIncomeNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTax_8bcf0927-ee20-4bb4-b4d2-3f38ef983dba" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EarningsPerShareBasic_a4c72ed7-7679-40e2-93a2-e801c549b2f1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EarningsPerShareBasic"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_EarningsPerShareBasic_a4c72ed7-7679-40e2-93a2-e801c549b2f1" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EarningsPerShareDiluted_1a88ae2f-c27a-49ca-9555-ea2b9757a74d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EarningsPerShareDiluted"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_EarningsPerShareDiluted_1a88ae2f-c27a-49ca-9555-ea2b9757a74d" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingBasic_1014c793-b0c6-48d3-9632-7293da102a70" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_WeightedAverageNumberOfSharesOutstandingBasic_1014c793-b0c6-48d3-9632-7293da102a70" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding_3553ff6c-1c21-44f3-9686-cfa8f9434438" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_9a8fda35-e339-4c23-a4d4-86b00aa949d4" xlink:to="loc_us-gaap_WeightedAverageNumberOfDilutedSharesOutstanding_3553ff6c-1c21-44f3-9686-cfa8f9434438" xlink:type="arc" order="12"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/UnauditedCondensedConsolidatedStatementsofChangesinShareholdersEquity" xlink:type="simple" xlink:href="autl-20250630.xsd#UnauditedCondensedConsolidatedStatementsofChangesinShareholdersEquity"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/UnauditedCondensedConsolidatedStatementsofChangesinShareholdersEquity" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_94f0e6b7-36da-49a1-adef-8299aea73c1c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_3797505f-9b3f-45e0-bb93-e35251b7cbdf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_94f0e6b7-36da-49a1-adef-8299aea73c1c" xlink:to="loc_us-gaap_StatementTable_3797505f-9b3f-45e0-bb93-e35251b7cbdf" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_fff786eb-3725-449e-9063-870d00ee8dd4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_3797505f-9b3f-45e0-bb93-e35251b7cbdf" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_fff786eb-3725-449e-9063-870d00ee8dd4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_fff786eb-3725-449e-9063-870d00ee8dd4_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_fff786eb-3725-449e-9063-870d00ee8dd4" xlink:to="loc_us-gaap_EquityComponentDomain_fff786eb-3725-449e-9063-870d00ee8dd4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_fff786eb-3725-449e-9063-870d00ee8dd4" xlink:to="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_64cd89b0-df1a-473e-ac21-ea36143fe1a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:to="loc_us-gaap_CommonStockMember_64cd89b0-df1a-473e-ac21-ea36143fe1a5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_88096ee3-35e8-40cb-9bf4-72255dbc2e81" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_88096ee3-35e8-40cb-9bf4-72255dbc2e81" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_88167535-8f93-43fb-9080-f0d2e8bd50e0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_88167535-8f93-43fb-9080-f0d2e8bd50e0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_a355e2a8-828d-46d3-b0b7-c1c2f65eee9f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_618be76b-6171-46d8-bdeb-d6173d59ddb2" xlink:to="loc_us-gaap_RetainedEarningsMember_a355e2a8-828d-46d3-b0b7-c1c2f65eee9f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_df79734e-933f-4293-8153-85ce5e202a27" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_3797505f-9b3f-45e0-bb93-e35251b7cbdf" xlink:to="loc_us-gaap_StatementClassOfStockAxis_df79734e-933f-4293-8153-85ce5e202a27" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_df79734e-933f-4293-8153-85ce5e202a27_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_df79734e-933f-4293-8153-85ce5e202a27" xlink:to="loc_us-gaap_ClassOfStockDomain_df79734e-933f-4293-8153-85ce5e202a27_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_df79734e-933f-4293-8153-85ce5e202a27" xlink:to="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_OrdinarySharesMember_95e07228-cfdc-48a8-87a7-c65ab4d180cd" xlink:href="autl-20250630.xsd#autl_OrdinarySharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:to="loc_autl_OrdinarySharesMember_95e07228-cfdc-48a8-87a7-c65ab4d180cd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredSharesMember_72d704a5-b700-4f2f-8d8c-9f4fde701b74" xlink:href="autl-20250630.xsd#autl_DeferredSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:to="loc_autl_DeferredSharesMember_72d704a5-b700-4f2f-8d8c-9f4fde701b74" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassBSharesMember_ab9e1169-e4f1-40d0-8912-b8f69b08b369" xlink:href="autl-20250630.xsd#autl_DeferredClassBSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:to="loc_autl_DeferredClassBSharesMember_ab9e1169-e4f1-40d0-8912-b8f69b08b369" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DeferredClassCSharesMember_c8ab7415-80b0-4346-b9fa-d8d713213910" xlink:href="autl-20250630.xsd#autl_DeferredClassCSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_a1a07e47-0226-4695-bb9e-c740faf30cb1" xlink:to="loc_autl_DeferredClassCSharesMember_c8ab7415-80b0-4346-b9fa-d8d713213910" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_94f0e6b7-36da-49a1-adef-8299aea73c1c" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_3dec1a7a-c3f5-479a-b724-e229ae5317bb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_3dec1a7a-c3f5-479a-b724-e229ae5317bb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_2acc99f0-35fc-417d-bb09-8861374d30cf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_StockholdersEquity_2acc99f0-35fc-417d-bb09-8861374d30cf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_a38764d2-957a-46be-96ba-85e5d45d1be0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_a38764d2-957a-46be-96ba-85e5d45d1be0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_0a9ce50c-c3ee-41a6-bce1-855f15bfe301" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_0a9ce50c-c3ee-41a6-bce1-855f15bfe301" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_09d3c619-2d59-41d0-b48a-de2c5e48e743" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_09d3c619-2d59-41d0-b48a-de2c5e48e743" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_StockVestedDuringPeriodSharesRestrictedStock_527aa3a4-74bc-49d6-844d-42cb538aa276" xlink:href="autl-20250630.xsd#autl_StockVestedDuringPeriodSharesRestrictedStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_autl_StockVestedDuringPeriodSharesRestrictedStock_527aa3a4-74bc-49d6-844d-42cb538aa276" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_78c87f43-6a1d-4710-911e-b62d64b47c87" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_78c87f43-6a1d-4710-911e-b62d64b47c87" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_d2b6334b-ad87-4be9-9a22-d1f09879afe2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_d2b6334b-ad87-4be9-9a22-d1f09879afe2" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTax_c0e110ca-6587-470f-8677-928cb0c64c52" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTax_c0e110ca-6587-470f-8677-928cb0c64c52" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_7b80bca0-faf0-4ead-b94d-0f0aee4ca4b5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_05508868-54f2-4d13-932a-23df6788bd64" xlink:to="loc_us-gaap_NetIncomeLoss_7b80bca0-faf0-4ead-b94d-0f0aee4ca4b5" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_5564f104-b6b6-4494-a481-4e447647b9ed" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_1bd9b96b-27e7-4506-a0ab-4b324a7f68c8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/RevenueScheduleofProductRevenueNetbyGeographicalLocationDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#RevenueScheduleofProductRevenueNetbyGeographicalLocationDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/RevenueScheduleofProductRevenueNetbyGeographicalLocationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_8ed90bb0-439a-42ec-8f19-2e3928674bb4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_89fb1c39-6990-48c5-a9f2-aaf37e8b3d8e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_8ed90bb0-439a-42ec-8f19-2e3928674bb4" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_89fb1c39-6990-48c5-a9f2-aaf37e8b3d8e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_1a4eb856-cf3e-4824-ade6-6b7a26703b42" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_89fb1c39-6990-48c5-a9f2-aaf37e8b3d8e" xlink:to="loc_srt_ProductOrServiceAxis_1a4eb856-cf3e-4824-ade6-6b7a26703b42" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_1a4eb856-cf3e-4824-ade6-6b7a26703b42_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_1a4eb856-cf3e-4824-ade6-6b7a26703b42" xlink:to="loc_srt_ProductsAndServicesDomain_1a4eb856-cf3e-4824-ade6-6b7a26703b42_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_a7dfb9a8-e3f6-4562-8367-13bbf3b3731e" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_1a4eb856-cf3e-4824-ade6-6b7a26703b42" xlink:to="loc_srt_ProductsAndServicesDomain_a7dfb9a8-e3f6-4562-8367-13bbf3b3731e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProductMember_eb2314d7-0096-488f-9d5c-e25a7fd7aeb8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProductMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_a7dfb9a8-e3f6-4562-8367-13bbf3b3731e" xlink:to="loc_us-gaap_ProductMember_eb2314d7-0096-488f-9d5c-e25a7fd7aeb8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_70695d8a-5083-462c-b618-f0e7f66b3f9e" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_89fb1c39-6990-48c5-a9f2-aaf37e8b3d8e" xlink:to="loc_srt_StatementGeographicalAxis_70695d8a-5083-462c-b618-f0e7f66b3f9e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_70695d8a-5083-462c-b618-f0e7f66b3f9e_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_70695d8a-5083-462c-b618-f0e7f66b3f9e" xlink:to="loc_srt_SegmentGeographicalDomain_70695d8a-5083-462c-b618-f0e7f66b3f9e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_cc66f4a1-b748-446b-9911-8df6dd7cffe3" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_70695d8a-5083-462c-b618-f0e7f66b3f9e" xlink:to="loc_srt_SegmentGeographicalDomain_cc66f4a1-b748-446b-9911-8df6dd7cffe3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_US_fe1f1a3f-f97b-49f6-9c8d-3e7d650dfb29" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_cc66f4a1-b748-446b-9911-8df6dd7cffe3" xlink:to="loc_country_US_fe1f1a3f-f97b-49f6-9c8d-3e7d650dfb29" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_5a7da3eb-3cdd-4e08-a329-9d0e0192e985" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_8ed90bb0-439a-42ec-8f19-2e3928674bb4" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_5a7da3eb-3cdd-4e08-a329-9d0e0192e985" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/RevenueNarrativeDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#RevenueNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/RevenueNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MajorCustomersAxis_2567e2fe-ef48-4193-84f2-d2f81245451a" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MajorCustomersAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:to="loc_srt_MajorCustomersAxis_2567e2fe-ef48-4193-84f2-d2f81245451a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_NameOfMajorCustomerDomain_2567e2fe-ef48-4193-84f2-d2f81245451a_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_NameOfMajorCustomerDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_MajorCustomersAxis_2567e2fe-ef48-4193-84f2-d2f81245451a" xlink:to="loc_srt_NameOfMajorCustomerDomain_2567e2fe-ef48-4193-84f2-d2f81245451a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_NameOfMajorCustomerDomain_926e0f74-0459-4ddf-baee-0f85471e788f" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_NameOfMajorCustomerDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_MajorCustomersAxis_2567e2fe-ef48-4193-84f2-d2f81245451a" xlink:to="loc_srt_NameOfMajorCustomerDomain_926e0f74-0459-4ddf-baee-0f85471e788f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CardinalHealth105LLCCardinalHealthMember_ae63f8ab-3849-419c-8feb-380702a6261d" xlink:href="autl-20250630.xsd#autl_CardinalHealth105LLCCardinalHealthMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_NameOfMajorCustomerDomain_926e0f74-0459-4ddf-baee-0f85471e788f" xlink:to="loc_autl_CardinalHealth105LLCCardinalHealthMember_ae63f8ab-3849-419c-8feb-380702a6261d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_8e988564-e8ba-4105-a9b2-043065bfeb12" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_NameOfMajorCustomerDomain_926e0f74-0459-4ddf-baee-0f85471e788f" xlink:to="loc_autl_BioNTechMember_8e988564-e8ba-4105-a9b2-043065bfeb12" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskByBenchmarkAxis_ba60b331-7a4d-4cca-b916-6b3bd0178e27" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskByBenchmarkAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:to="loc_us-gaap_ConcentrationRiskByBenchmarkAxis_ba60b331-7a4d-4cca-b916-6b3bd0178e27" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskBenchmarkDomain_ba60b331-7a4d-4cca-b916-6b3bd0178e27_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskBenchmarkDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ConcentrationRiskByBenchmarkAxis_ba60b331-7a4d-4cca-b916-6b3bd0178e27" xlink:to="loc_us-gaap_ConcentrationRiskBenchmarkDomain_ba60b331-7a4d-4cca-b916-6b3bd0178e27_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskBenchmarkDomain_e932f58c-13d4-4ea1-ac50-6dc2940b434e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskBenchmarkDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ConcentrationRiskByBenchmarkAxis_ba60b331-7a4d-4cca-b916-6b3bd0178e27" xlink:to="loc_us-gaap_ConcentrationRiskBenchmarkDomain_e932f58c-13d4-4ea1-ac50-6dc2940b434e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SalesRevenueNetMember_4ab755bd-1ea2-420f-b808-6c02ef4c30fc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SalesRevenueNetMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ConcentrationRiskBenchmarkDomain_e932f58c-13d4-4ea1-ac50-6dc2940b434e" xlink:to="loc_us-gaap_SalesRevenueNetMember_4ab755bd-1ea2-420f-b808-6c02ef4c30fc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskByTypeAxis_06bf793c-a858-4743-b42a-9997aaa45c3d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:to="loc_us-gaap_ConcentrationRiskByTypeAxis_06bf793c-a858-4743-b42a-9997aaa45c3d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskTypeDomain_06bf793c-a858-4743-b42a-9997aaa45c3d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ConcentrationRiskByTypeAxis_06bf793c-a858-4743-b42a-9997aaa45c3d" xlink:to="loc_us-gaap_ConcentrationRiskTypeDomain_06bf793c-a858-4743-b42a-9997aaa45c3d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskTypeDomain_6aa0771e-ce01-4ade-be66-2f3d7b7f8a7d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ConcentrationRiskByTypeAxis_06bf793c-a858-4743-b42a-9997aaa45c3d" xlink:to="loc_us-gaap_ConcentrationRiskTypeDomain_6aa0771e-ce01-4ade-be66-2f3d7b7f8a7d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CustomerConcentrationRiskMember_a3ada33f-de17-4fd2-ad2e-726e0f2e9c2f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CustomerConcentrationRiskMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ConcentrationRiskTypeDomain_6aa0771e-ce01-4ade-be66-2f3d7b7f8a7d" xlink:to="loc_us-gaap_CustomerConcentrationRiskMember_a3ada33f-de17-4fd2-ad2e-726e0f2e9c2f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_079f65b7-44b5-44eb-b4cf-8e92bc462b1a" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:to="loc_srt_ProductOrServiceAxis_079f65b7-44b5-44eb-b4cf-8e92bc462b1a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_079f65b7-44b5-44eb-b4cf-8e92bc462b1a_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_079f65b7-44b5-44eb-b4cf-8e92bc462b1a" xlink:to="loc_srt_ProductsAndServicesDomain_079f65b7-44b5-44eb-b4cf-8e92bc462b1a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_8e2f2258-6aac-4921-b41b-c958c2f173a2" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_079f65b7-44b5-44eb-b4cf-8e92bc462b1a" xlink:to="loc_srt_ProductsAndServicesDomain_8e2f2258-6aac-4921-b41b-c958c2f173a2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicenseMember_79b9db8e-647a-4939-a7fb-90106dd702a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LicenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_8e2f2258-6aac-4921-b41b-c958c2f173a2" xlink:to="loc_us-gaap_LicenseMember_79b9db8e-647a-4939-a7fb-90106dd702a5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_LicenseAndOptionsMember_cbcf8ef5-19b2-4a2c-a822-1610ef660f6e" xlink:href="autl-20250630.xsd#autl_LicenseAndOptionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_8e2f2258-6aac-4921-b41b-c958c2f173a2" xlink:to="loc_autl_LicenseAndOptionsMember_cbcf8ef5-19b2-4a2c-a822-1610ef660f6e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_TechnologyOptionsMember_c24dff6e-c0c1-465e-8105-f4a6b63b0c45" xlink:href="autl-20250630.xsd#autl_TechnologyOptionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_8e2f2258-6aac-4921-b41b-c958c2f173a2" xlink:to="loc_autl_TechnologyOptionsMember_c24dff6e-c0c1-465e-8105-f4a6b63b0c45" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_a5be55a7-1f53-4aad-9cf4-611b4563efd9" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_4d756c04-d8e5-44bd-8176-6a82fbd47c0a" xlink:to="loc_srt_CounterpartyNameAxis_a5be55a7-1f53-4aad-9cf4-611b4563efd9" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_a5be55a7-1f53-4aad-9cf4-611b4563efd9_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_a5be55a7-1f53-4aad-9cf4-611b4563efd9" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_a5be55a7-1f53-4aad-9cf4-611b4563efd9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_fb425bc6-4547-4583-a173-11a12f366003" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_a5be55a7-1f53-4aad-9cf4-611b4563efd9" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_fb425bc6-4547-4583-a173-11a12f366003" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_f069b8b2-5a1d-4d7d-bca0-5da26de68d04" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_fb425bc6-4547-4583-a173-11a12f366003" xlink:to="loc_autl_BioNTechMember_f069b8b2-5a1d-4d7d-bca0-5da26de68d04" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConcentrationRiskPercentage1_90dab42e-cdf3-4c9e-8a4b-e37b2be7af18" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskPercentage1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_us-gaap_ConcentrationRiskPercentage1_90dab42e-cdf3-4c9e-8a4b-e37b2be7af18" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsAndOtherReceivablesNetCurrent_d997bc65-b1d5-4069-a229-aaecdf0f9633" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccountsAndOtherReceivablesNetCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_us-gaap_AccountsAndOtherReceivablesNetCurrent_d997bc65-b1d5-4069-a229-aaecdf0f9633" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_AccountsAndOtherReceivablesNetCurrentRounding_e47607b2-06f2-4ca3-ad10-43075f2c4ae5" xlink:href="autl-20250630.xsd#autl_AccountsAndOtherReceivablesNetCurrentRounding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_autl_AccountsAndOtherReceivablesNetCurrentRounding_e47607b2-06f2-4ca3-ad10-43075f2c4ae5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_NumberOfFreestandingFinancialInstruments_3d86b90c-5b9b-44ca-b891-16cd900f7e52" xlink:href="autl-20250630.xsd#autl_NumberOfFreestandingFinancialInstruments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_autl_NumberOfFreestandingFinancialInstruments_3d86b90c-5b9b-44ca-b891-16cd900f7e52" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_NumberOfCombinedPerformanceObligations_76c68ab2-d370-4ac3-a3b7-ca0cf0bded1c" xlink:href="autl-20250630.xsd#autl_NumberOfCombinedPerformanceObligations"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_autl_NumberOfCombinedPerformanceObligations_76c68ab2-d370-4ac3-a3b7-ca0cf0bded1c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_81b61aa9-19e3-45c3-9116-62bbb5ca4fb6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_81b61aa9-19e3-45c3-9116-62bbb5ca4fb6" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount_7f95bea9-2b0d-4762-a5e2-dafac8164a45" xlink:href="autl-20250630.xsd#autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_c07fbff7-fa86-48a4-a2f1-9bc6d5942a14" xlink:to="loc_autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount_7f95bea9-2b0d-4762-a5e2-dafac8164a45" xlink:type="arc" order="6"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/RevenueScheduleofCurrentAccrualsforRebatesandChargebacksDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#RevenueScheduleofCurrentAccrualsforRebatesandChargebacksDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/RevenueScheduleofCurrentAccrualsforRebatesandChargebacksDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_9f660adf-3ae2-43fb-ad72-c37edc69ab52" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_b4fde0b1-1ea5-4f67-9f00-5dcd78b20a4c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_9f660adf-3ae2-43fb-ad72-c37edc69ab52" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_b4fde0b1-1ea5-4f67-9f00-5dcd78b20a4c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_b4fde0b1-1ea5-4f67-9f00-5dcd78b20a4c" xlink:to="loc_srt_ProductOrServiceAxis_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6" xlink:to="loc_srt_ProductsAndServicesDomain_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_6c77605d-c2a0-43f7-b047-2732350a2fb4" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_e15d2a1a-8c9f-4a39-96c7-fa0d08683fb6" xlink:to="loc_srt_ProductsAndServicesDomain_6c77605d-c2a0-43f7-b047-2732350a2fb4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RebatesMember_946c4fa5-c241-4fbd-91ab-db17d1940fd3" xlink:href="autl-20250630.xsd#autl_RebatesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_6c77605d-c2a0-43f7-b047-2732350a2fb4" xlink:to="loc_autl_RebatesMember_946c4fa5-c241-4fbd-91ab-db17d1940fd3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ChargebackMember_a050173e-7e96-4cb2-8767-2a1e977b3999" xlink:href="autl-20250630.xsd#autl_ChargebackMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_6c77605d-c2a0-43f7-b047-2732350a2fb4" xlink:to="loc_autl_ChargebackMember_a050173e-7e96-4cb2-8767-2a1e977b3999" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward_74d34e89-1b51-4686-8709-b093beea44c8" xlink:href="autl-20250630.xsd#autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_9f660adf-3ae2-43fb-ad72-c37edc69ab52" xlink:to="loc_autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward_74d34e89-1b51-4686-8709-b093beea44c8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerRefundLiabilityCurrent_34055030-30ea-40d1-a7b5-4cd835022b62" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerRefundLiabilityCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward_74d34e89-1b51-4686-8709-b093beea44c8" xlink:to="loc_us-gaap_ContractWithCustomerRefundLiabilityCurrent_34055030-30ea-40d1-a7b5-4cd835022b62" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_IncreaseDecreaseInContractWithCustomerRefundLiabilitySales_be8bac2e-2bab-4430-b401-9337f2712443" xlink:href="autl-20250630.xsd#autl_IncreaseDecreaseInContractWithCustomerRefundLiabilitySales"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward_74d34e89-1b51-4686-8709-b093beea44c8" xlink:to="loc_autl_IncreaseDecreaseInContractWithCustomerRefundLiabilitySales_be8bac2e-2bab-4430-b401-9337f2712443" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractWithCustomerRefundLiabilityPaymentsAndCredits_a5cf00bd-7fbb-4b14-a490-2a68609c1dfa" xlink:href="autl-20250630.xsd#autl_ContractWithCustomerRefundLiabilityPaymentsAndCredits"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_autl_CustomerRefundLiabilityCurrentAndNonCurrentRollForward_74d34e89-1b51-4686-8709-b093beea44c8" xlink:to="loc_autl_ContractWithCustomerRefundLiabilityPaymentsAndCredits_a5cf00bd-7fbb-4b14-a490-2a68609c1dfa" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerRefundLiabilityCurrent_71237442-e040-4f49-b6b8-aefa8044975a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractWithCustomerRefundLiabilityCurrent"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/RevenueScheduleofLicenseRevenuebyGeographicalLocationDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#RevenueScheduleofLicenseRevenuebyGeographicalLocationDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/RevenueScheduleofLicenseRevenuebyGeographicalLocationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_8a6cf967-937f-4c8b-81bd-b7c9edc49e2a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_27af1102-f19e-4ce3-a906-dfe09f7e2ad1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_8a6cf967-937f-4c8b-81bd-b7c9edc49e2a" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_27af1102-f19e-4ce3-a906-dfe09f7e2ad1" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_961ab51a-3340-49f7-9d01-d2b74720bf69" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_27af1102-f19e-4ce3-a906-dfe09f7e2ad1" xlink:to="loc_srt_ProductOrServiceAxis_961ab51a-3340-49f7-9d01-d2b74720bf69" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_961ab51a-3340-49f7-9d01-d2b74720bf69_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_961ab51a-3340-49f7-9d01-d2b74720bf69" xlink:to="loc_srt_ProductsAndServicesDomain_961ab51a-3340-49f7-9d01-d2b74720bf69_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_6dcfdfc2-4668-4e87-ad88-31a177927198" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_961ab51a-3340-49f7-9d01-d2b74720bf69" xlink:to="loc_srt_ProductsAndServicesDomain_6dcfdfc2-4668-4e87-ad88-31a177927198" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicenseMember_997f2bf2-3559-4b11-850b-74adf8493ded" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LicenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_6dcfdfc2-4668-4e87-ad88-31a177927198" xlink:to="loc_us-gaap_LicenseMember_997f2bf2-3559-4b11-850b-74adf8493ded" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_80d72813-dc36-4534-9e55-533d1b16a28e" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_27af1102-f19e-4ce3-a906-dfe09f7e2ad1" xlink:to="loc_srt_StatementGeographicalAxis_80d72813-dc36-4534-9e55-533d1b16a28e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_80d72813-dc36-4534-9e55-533d1b16a28e_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_80d72813-dc36-4534-9e55-533d1b16a28e" xlink:to="loc_srt_SegmentGeographicalDomain_80d72813-dc36-4534-9e55-533d1b16a28e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_15f32b8c-bd92-4b49-96ab-0a4d832dceef" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_80d72813-dc36-4534-9e55-533d1b16a28e" xlink:to="loc_srt_SegmentGeographicalDomain_15f32b8c-bd92-4b49-96ab-0a4d832dceef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_EuropeMember_58802e3d-4d80-4538-ba77-5034e32f894b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_EuropeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_15f32b8c-bd92-4b49-96ab-0a4d832dceef" xlink:to="loc_srt_EuropeMember_58802e3d-4d80-4538-ba77-5034e32f894b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_d78b3daa-db45-4bc4-b4d8-743cda9257b2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_8a6cf967-937f-4c8b-81bd-b7c9edc49e2a" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_d78b3daa-db45-4bc4-b4d8-743cda9257b2" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/NetLossPerShareScheduleofPotentiallyDilutiveSecuritiesExcludedfromComputationofDilutedNetLossPerShareDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#NetLossPerShareScheduleofPotentiallyDilutiveSecuritiesExcludedfromComputationofDilutedNetLossPerShareDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/NetLossPerShareScheduleofPotentiallyDilutiveSecuritiesExcludedfromComputationofDilutedNetLossPerShareDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_890a5430-081d-49e5-8470-b07353f105ee" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f3f9d30a-2471-40ad-93f8-d2ac9dbb984d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_890a5430-081d-49e5-8470-b07353f105ee" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f3f9d30a-2471-40ad-93f8-d2ac9dbb984d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_f3f9d30a-2471-40ad-93f8-d2ac9dbb984d" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_7a1560d0-329d-48b8-8105-aa297bc4246f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_76b5a4e8-7de1-4a5a-9908-095f1bdbc1e1" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_7a1560d0-329d-48b8-8105-aa297bc4246f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_6a3961d3-6d24-4f56-925a-fb1f83d93649" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_7a1560d0-329d-48b8-8105-aa297bc4246f" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_6a3961d3-6d24-4f56-925a-fb1f83d93649" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_93d18dc0-b1ae-426b-b341-f29486e7897c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_7a1560d0-329d-48b8-8105-aa297bc4246f" xlink:to="loc_us-gaap_EmployeeStockOptionMember_93d18dc0-b1ae-426b-b341-f29486e7897c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_c2f455a4-71d6-4f1a-b301-748757148268" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_7a1560d0-329d-48b8-8105-aa297bc4246f" xlink:to="loc_us-gaap_WarrantMember_c2f455a4-71d6-4f1a-b301-748757148268" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_30b7bc8f-3708-43fa-891b-3514f9cf73b6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_890a5430-081d-49e5-8470-b07353f105ee" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_30b7bc8f-3708-43fa-891b-3514f9cf73b6" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/FairValueMeasurementsDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#FairValueMeasurementsDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/FairValueMeasurementsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_fdb02b69-1859-43ee-b8d5-aaeeedd3c9e0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_0d6f2450-a075-4584-b871-f5b01aae7431" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_fdb02b69-1859-43ee-b8d5-aaeeedd3c9e0" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_0d6f2450-a075-4584-b871-f5b01aae7431" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsAxis_0b501191-bae8-4839-b8b3-3382da145ee5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_0d6f2450-a075-4584-b871-f5b01aae7431" xlink:to="loc_us-gaap_CashAndCashEquivalentsAxis_0b501191-bae8-4839-b8b3-3382da145ee5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_0b501191-bae8-4839-b8b3-3382da145ee5_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CashAndCashEquivalentsAxis_0b501191-bae8-4839-b8b3-3382da145ee5" xlink:to="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_0b501191-bae8-4839-b8b3-3382da145ee5_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CashAndCashEquivalentsAxis_0b501191-bae8-4839-b8b3-3382da145ee5" xlink:to="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MoneyMarketFundsMember_9c8282f9-9416-4979-becc-5262a8729c7d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MoneyMarketFundsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:to="loc_us-gaap_MoneyMarketFundsMember_9c8282f9-9416-4979-becc-5262a8729c7d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_517c33a8-64fe-463b-b739-b06b9ffa9cd3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:to="loc_us-gaap_CommercialPaperMember_517c33a8-64fe-463b-b739-b06b9ffa9cd3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_61d8b646-4207-4f5a-85a5-05cf40406a18" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignGovernmentDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:to="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_61d8b646-4207-4f5a-85a5-05cf40406a18" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryBillSecuritiesMember_fdafb6ab-57dd-4381-9e87-a4db04dcffcb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasuryBillSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_28df04cc-7713-49ab-9ee1-b72911242dce" xlink:to="loc_us-gaap_USTreasuryBillSecuritiesMember_fdafb6ab-57dd-4381-9e87-a4db04dcffcb" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_0d6f2450-a075-4584-b871-f5b01aae7431" xlink:to="loc_us-gaap_FinancialInstrumentAxis_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_5b9ac27e-3e82-47cd-b63f-9ef4cea571cd" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_1ebfedf6-7a0b-4de8-bf54-c955a356542d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:to="loc_us-gaap_CommercialPaperMember_1ebfedf6-7a0b-4de8-bf54-c955a356542d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateDebtSecuritiesMember_cb7cb550-916b-4163-8d2c-4bf2ea226ca2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:to="loc_us-gaap_CorporateDebtSecuritiesMember_cb7cb550-916b-4163-8d2c-4bf2ea226ca2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_bc079e50-6b0a-4177-b5e5-e9ba4fd14388" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignGovernmentDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:to="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_bc079e50-6b0a-4177-b5e5-e9ba4fd14388" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryBillSecuritiesMember_18af28fe-9085-480e-9914-2e8d43e26247" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasuryBillSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_98614cfa-14c2-441c-b3a5-f8755413dfa4" xlink:to="loc_us-gaap_USTreasuryBillSecuritiesMember_18af28fe-9085-480e-9914-2e8d43e26247" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_d6e9038f-c522-4cb1-a486-a1005aeecc88" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_0d6f2450-a075-4584-b871-f5b01aae7431" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_d6e9038f-c522-4cb1-a486-a1005aeecc88" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_d6e9038f-c522-4cb1-a486-a1005aeecc88_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_d6e9038f-c522-4cb1-a486-a1005aeecc88" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_d6e9038f-c522-4cb1-a486-a1005aeecc88_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3a3707d6-c199-46e1-9a2d-98a9a277c981" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_d6e9038f-c522-4cb1-a486-a1005aeecc88" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3a3707d6-c199-46e1-9a2d-98a9a277c981" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_46c64c63-9eb3-4343-a01b-5069c71e6bd5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3a3707d6-c199-46e1-9a2d-98a9a277c981" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_46c64c63-9eb3-4343-a01b-5069c71e6bd5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_51fa2cab-63c3-4089-97e9-c894f4c98ed8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3a3707d6-c199-46e1-9a2d-98a9a277c981" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_51fa2cab-63c3-4089-97e9-c894f4c98ed8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel3Member_80a2ff41-e4f7-4003-8ef6-854000044acb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel3Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3a3707d6-c199-46e1-9a2d-98a9a277c981" xlink:to="loc_us-gaap_FairValueInputsLevel3Member_80a2ff41-e4f7-4003-8ef6-854000044acb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_eb0d0170-d067-4e77-bf05-f9499030c40d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_fdb02b69-1859-43ee-b8d5-aaeeedd3c9e0" xlink:to="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_eb0d0170-d067-4e77-bf05-f9499030c40d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_03d174f4-9a49-4fe5-a3cf-a616f4019a8f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_fdb02b69-1859-43ee-b8d5-aaeeedd3c9e0" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_03d174f4-9a49-4fe5-a3cf-a616f4019a8f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsFairValueDisclosure_d10a4610-eb50-4864-b06f-c2cf0979fb00" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_fdb02b69-1859-43ee-b8d5-aaeeedd3c9e0" xlink:to="loc_us-gaap_AssetsFairValueDisclosure_d10a4610-eb50-4864-b06f-c2cf0979fb00" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofInvestmentsinAvailableforSaleDebtSecuritiesDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofInvestmentsinAvailableforSaleDebtSecuritiesDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofInvestmentsinAvailableforSaleDebtSecuritiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleTable_566e6bb8-47dd-42cf-b5b2-7d66530a05f7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleTable_566e6bb8-47dd-42cf-b5b2-7d66530a05f7" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractualMaturityAxis_0194c856-d108-403a-b802-1e2befac8451" xlink:href="autl-20250630.xsd#autl_ContractualMaturityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleTable_566e6bb8-47dd-42cf-b5b2-7d66530a05f7" xlink:to="loc_autl_ContractualMaturityAxis_0194c856-d108-403a-b802-1e2befac8451" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractualMaturityDomain_0194c856-d108-403a-b802-1e2befac8451_default" xlink:href="autl-20250630.xsd#autl_ContractualMaturityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_autl_ContractualMaturityAxis_0194c856-d108-403a-b802-1e2befac8451" xlink:to="loc_autl_ContractualMaturityDomain_0194c856-d108-403a-b802-1e2befac8451_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractualMaturityDomain_777483dd-b7cb-44b5-95ca-4f41b20b8ca1" xlink:href="autl-20250630.xsd#autl_ContractualMaturityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_autl_ContractualMaturityAxis_0194c856-d108-403a-b802-1e2befac8451" xlink:to="loc_autl_ContractualMaturityDomain_777483dd-b7cb-44b5-95ca-4f41b20b8ca1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractualMaturityWithinOneYearMember_705f76ee-e36a-49d2-bf9c-a9599f9afe03" xlink:href="autl-20250630.xsd#autl_ContractualMaturityWithinOneYearMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_autl_ContractualMaturityDomain_777483dd-b7cb-44b5-95ca-4f41b20b8ca1" xlink:to="loc_autl_ContractualMaturityWithinOneYearMember_705f76ee-e36a-49d2-bf9c-a9599f9afe03" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ContractualMaturityWithinOneThroughFiveYearsMember_c66b14ae-26e5-4789-be8d-7fb6ccc087c1" xlink:href="autl-20250630.xsd#autl_ContractualMaturityWithinOneThroughFiveYearsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_autl_ContractualMaturityDomain_777483dd-b7cb-44b5-95ca-4f41b20b8ca1" xlink:to="loc_autl_ContractualMaturityWithinOneThroughFiveYearsMember_c66b14ae-26e5-4789-be8d-7fb6ccc087c1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_dcc32257-8e18-4021-80a9-c64ff020f77f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleTable_566e6bb8-47dd-42cf-b5b2-7d66530a05f7" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_dcc32257-8e18-4021-80a9-c64ff020f77f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_dcc32257-8e18-4021-80a9-c64ff020f77f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_dcc32257-8e18-4021-80a9-c64ff020f77f" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_dcc32257-8e18-4021-80a9-c64ff020f77f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_daffe2de-3f4e-4d1f-b791-cfe28684a5d6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_dcc32257-8e18-4021-80a9-c64ff020f77f" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_daffe2de-3f4e-4d1f-b791-cfe28684a5d6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DebtSecuritiesAvailableForSaleMember_c495f758-e78d-47d5-9728-065eae0daf69" xlink:href="autl-20250630.xsd#autl_DebtSecuritiesAvailableForSaleMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_daffe2de-3f4e-4d1f-b791-cfe28684a5d6" xlink:to="loc_autl_DebtSecuritiesAvailableForSaleMember_c495f758-e78d-47d5-9728-065eae0daf69" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_88664e01-bda4-4aaf-b513-84d70a569f57" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleTable_566e6bb8-47dd-42cf-b5b2-7d66530a05f7" xlink:to="loc_us-gaap_FinancialInstrumentAxis_88664e01-bda4-4aaf-b513-84d70a569f57" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_88664e01-bda4-4aaf-b513-84d70a569f57_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_88664e01-bda4-4aaf-b513-84d70a569f57" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_88664e01-bda4-4aaf-b513-84d70a569f57_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_88664e01-bda4-4aaf-b513-84d70a569f57" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_cd7ffcd4-1da8-4b2b-8b8e-8704fb8573b7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:to="loc_us-gaap_CommercialPaperMember_cd7ffcd4-1da8-4b2b-8b8e-8704fb8573b7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateDebtSecuritiesMember_6c8368fc-20da-4865-ad97-8348cae403f6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:to="loc_us-gaap_CorporateDebtSecuritiesMember_6c8368fc-20da-4865-ad97-8348cae403f6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_3398f7b1-69e3-48d0-8260-09b710610ab2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignGovernmentDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:to="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_3398f7b1-69e3-48d0-8260-09b710610ab2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryBillSecuritiesMember_75e8d850-a053-45f7-ab8c-b01fe2a26f06" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasuryBillSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_8ddd0ec8-bc2d-4fad-b7d3-d3ec60064830" xlink:to="loc_us-gaap_USTreasuryBillSecuritiesMember_75e8d850-a053-45f7-ab8c-b01fe2a26f06" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_58731dc0-6763-4aad-8968-de060a27ab82" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_58731dc0-6763-4aad-8968-de060a27ab82" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_c742bff5-681f-4c2f-a1e7-0e19a3fbf143" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax_c742bff5-681f-4c2f-a1e7-0e19a3fbf143" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_7da93aef-86e8-40e1-9bcd-64ae1973368e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax_7da93aef-86e8-40e1-9bcd-64ae1973368e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_06e44e36-34f8-4fc7-afc3-8ce23acc3edb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems_befd2b4a-8f17-4d42-b808-592f10064288" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_06e44e36-34f8-4fc7-afc3-8ce23acc3edb" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofNumberofSecuritiesHeldbyCompanyAggregateFairValueandUnrealizedLossPositionDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofNumberofSecuritiesHeldbyCompanyAggregateFairValueandUnrealizedLossPositionDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesScheduleofNumberofSecuritiesHeldbyCompanyAggregateFairValueandUnrealizedLossPositionDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_69f80bed-5551-47ce-8e02-9662311e586d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_1d0bcde3-5daa-4b84-9688-0ab083ad225d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_69f80bed-5551-47ce-8e02-9662311e586d" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_1d0bcde3-5daa-4b84-9688-0ab083ad225d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_8c0529b0-2b7b-4ed9-97ab-a53cf649029e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_1d0bcde3-5daa-4b84-9688-0ab083ad225d" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_8c0529b0-2b7b-4ed9-97ab-a53cf649029e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_8c0529b0-2b7b-4ed9-97ab-a53cf649029e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_8c0529b0-2b7b-4ed9-97ab-a53cf649029e" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_8c0529b0-2b7b-4ed9-97ab-a53cf649029e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_6cdac506-d5a1-4cc9-8922-fd47afed9cbd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_8c0529b0-2b7b-4ed9-97ab-a53cf649029e" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_6cdac506-d5a1-4cc9-8922-fd47afed9cbd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DebtSecuritiesAvailableForSaleMember_da6ad378-41de-44be-a316-60db0700aab0" xlink:href="autl-20250630.xsd#autl_DebtSecuritiesAvailableForSaleMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_6cdac506-d5a1-4cc9-8922-fd47afed9cbd" xlink:to="loc_autl_DebtSecuritiesAvailableForSaleMember_da6ad378-41de-44be-a316-60db0700aab0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_dfbc0755-d369-4a48-bf84-1dab6ce88b9c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_1d0bcde3-5daa-4b84-9688-0ab083ad225d" xlink:to="loc_us-gaap_FinancialInstrumentAxis_dfbc0755-d369-4a48-bf84-1dab6ce88b9c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_dfbc0755-d369-4a48-bf84-1dab6ce88b9c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_dfbc0755-d369-4a48-bf84-1dab6ce88b9c" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_dfbc0755-d369-4a48-bf84-1dab6ce88b9c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_dfbc0755-d369-4a48-bf84-1dab6ce88b9c" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_5aba0cfe-a84d-4af0-be54-458bdcb46f85" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:to="loc_us-gaap_CommercialPaperMember_5aba0cfe-a84d-4af0-be54-458bdcb46f85" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateDebtSecuritiesMember_de891603-bf7a-4716-b773-e0f78d8bc0de" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:to="loc_us-gaap_CorporateDebtSecuritiesMember_de891603-bf7a-4716-b773-e0f78d8bc0de" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_8af891d4-1711-4fd8-8613-22b05efbccdc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignGovernmentDebtSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:to="loc_us-gaap_ForeignGovernmentDebtSecuritiesMember_8af891d4-1711-4fd8-8613-22b05efbccdc" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryBillSecuritiesMember_2cc7fb49-82ed-41e5-b6b2-50d972bceddf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasuryBillSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_f75fdbe0-7d90-4c21-bad0-a65226f9c33b" xlink:to="loc_us-gaap_USTreasuryBillSecuritiesMember_2cc7fb49-82ed-41e5-b6b2-50d972bceddf" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions_188e0ebc-ef95-4987-878a-51ff57eb3968" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_69f80bed-5551-47ce-8e02-9662311e586d" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions_188e0ebc-ef95-4987-878a-51ff57eb3968" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_a4b5e3c6-3f0a-4569-9252-338408c9ecc9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_69f80bed-5551-47ce-8e02-9662311e586d" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_a4b5e3c6-3f0a-4569-9252-338408c9ecc9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition_a9f773fc-79a8-4980-8a28-b467cc31662b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_69f80bed-5551-47ce-8e02-9662311e586d" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition_a9f773fc-79a8-4980-8a28-b467cc31662b" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesNarrativeDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#MarketableSecuritiesAvailableForSaleDebtSecuritiesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/MarketableSecuritiesAvailableForSaleDebtSecuritiesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_5c8805df-5c7d-4281-bd7d-40e1e44219c4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_d1f5834c-23e6-4737-8be2-296d0bd68f7f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_5c8805df-5c7d-4281-bd7d-40e1e44219c4" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_d1f5834c-23e6-4737-8be2-296d0bd68f7f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_205ff317-108d-4a83-98f0-4177d15b745a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionFairValueTable_d1f5834c-23e6-4737-8be2-296d0bd68f7f" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_205ff317-108d-4a83-98f0-4177d15b745a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_205ff317-108d-4a83-98f0-4177d15b745a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_205ff317-108d-4a83-98f0-4177d15b745a" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_205ff317-108d-4a83-98f0-4177d15b745a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_7e3ea9a3-f2f2-429b-88ea-85d71a00d556" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_205ff317-108d-4a83-98f0-4177d15b745a" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_7e3ea9a3-f2f2-429b-88ea-85d71a00d556" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DebtSecuritiesAvailableForSaleMember_8a4ad760-efb0-40a3-99b8-6b361fc44562" xlink:href="autl-20250630.xsd#autl_DebtSecuritiesAvailableForSaleMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_7e3ea9a3-f2f2-429b-88ea-85d71a00d556" xlink:to="loc_autl_DebtSecuritiesAvailableForSaleMember_8a4ad760-efb0-40a3-99b8-6b361fc44562" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_35f39903-9dfc-4181-a866-258d04078cea" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_5c8805df-5c7d-4281-bd7d-40e1e44219c4" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss_35f39903-9dfc-4181-a866-258d04078cea" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions_ae44ce89-358f-4ee9-9bad-1c857ebd01e2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AvailableForSaleSecuritiesContinuousUnrealizedLossPositionFairValueAbstract_5c8805df-5c7d-4281-bd7d-40e1e44219c4" xlink:to="loc_us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsNumberOfPositions_ae44ce89-358f-4ee9-9bad-1c857ebd01e2" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/PropertyandEquipmentNetScheduleofPropertyandEquipmentNetDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#PropertyandEquipmentNetScheduleofPropertyandEquipmentNetDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/PropertyandEquipmentNetScheduleofPropertyandEquipmentNetDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentLineItems_e28069bd-8589-4cf6-8169-10097d0e47d8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_2db79439-2a2f-40cb-b0b7-5e9b6948d7d6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_e28069bd-8589-4cf6-8169-10097d0e47d8" xlink:to="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_2db79439-2a2f-40cb-b0b7-5e9b6948d7d6" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_fabdf834-5597-4806-820a-aa17f3883ee5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_2db79439-2a2f-40cb-b0b7-5e9b6948d7d6" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_fabdf834-5597-4806-820a-aa17f3883ee5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_fabdf834-5597-4806-820a-aa17f3883ee5_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_fabdf834-5597-4806-820a-aa17f3883ee5" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_fabdf834-5597-4806-820a-aa17f3883ee5_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_fabdf834-5597-4806-820a-aa17f3883ee5" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquipmentMember_5de62f4a-310c-4618-bbab-728421cce0cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:to="loc_us-gaap_EquipmentMember_5de62f4a-310c-4618-bbab-728421cce0cb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OfficeEquipmentMember_c9e70406-b2c4-4aa8-a5e0-f49ea124a67e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OfficeEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:to="loc_us-gaap_OfficeEquipmentMember_c9e70406-b2c4-4aa8-a5e0-f49ea124a67e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FurnitureAndFixturesMember_e0aae254-9702-4da1-a66e-2546b9e6998f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:to="loc_us-gaap_FurnitureAndFixturesMember_e0aae254-9702-4da1-a66e-2546b9e6998f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LeaseholdImprovementsMember_ba50289b-b1e3-4186-8043-f18fe8797699" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:to="loc_us-gaap_LeaseholdImprovementsMember_ba50289b-b1e3-4186-8043-f18fe8797699" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetUnderConstructionMember_c08ce7b1-993c-4aa2-a6f1-44a96f30bdb5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetUnderConstructionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_697a4008-653c-401a-a30f-51dbdd34b2f2" xlink:to="loc_us-gaap_AssetUnderConstructionMember_c08ce7b1-993c-4aa2-a6f1-44a96f30bdb5" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentGross_29822f2a-dd86-47a2-8b6b-a3c51087c56c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_e28069bd-8589-4cf6-8169-10097d0e47d8" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentGross_29822f2a-dd86-47a2-8b6b-a3c51087c56c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_423b48a4-2ccd-44c4-b319-31d3df3e54e3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_e28069bd-8589-4cf6-8169-10097d0e47d8" xlink:to="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_423b48a4-2ccd-44c4-b319-31d3df3e54e3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentNet_aa61f6e9-4fb7-4213-b564-9b2db0ba7e4b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_e28069bd-8589-4cf6-8169-10097d0e47d8" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentNet_aa61f6e9-4fb7-4213-b564-9b2db0ba7e4b" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/IntangibleAssetsNetScheduleofCarryingAmountofIntangibleAssetsNetofAccumulatedAmortizationDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#IntangibleAssetsNetScheduleofCarryingAmountofIntangibleAssetsNetofAccumulatedAmortizationDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/IntangibleAssetsNetScheduleofCarryingAmountofIntangibleAssetsNetofAccumulatedAmortizationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsLineItems_038692ff-6db8-445c-beff-df3c48d259a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable_d0c0c74e-3e51-464c-baa7-37d405af7f92" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsLineItems_038692ff-6db8-445c-beff-df3c48d259a5" xlink:to="loc_us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable_d0c0c74e-3e51-464c-baa7-37d405af7f92" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_709b2a2a-76cc-47c5-979d-073e00bdacc3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable_d0c0c74e-3e51-464c-baa7-37d405af7f92" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_709b2a2a-76cc-47c5-979d-073e00bdacc3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_709b2a2a-76cc-47c5-979d-073e00bdacc3_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_709b2a2a-76cc-47c5-979d-073e00bdacc3" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_709b2a2a-76cc-47c5-979d-073e00bdacc3_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_02409a6b-1649-4307-9ea4-e99acc8f9ea8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_709b2a2a-76cc-47c5-979d-073e00bdacc3" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_02409a6b-1649-4307-9ea4-e99acc8f9ea8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IntellectualPropertyMember_e3b0aeb4-c94e-4cd0-8b1b-0d943cf532b4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IntellectualPropertyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_02409a6b-1649-4307-9ea4-e99acc8f9ea8" xlink:to="loc_us-gaap_IntellectualPropertyMember_e3b0aeb4-c94e-4cd0-8b1b-0d943cf532b4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicensingAgreementsMember_b349a6d5-38fc-408c-876e-f6d417952995" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LicensingAgreementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_02409a6b-1649-4307-9ea4-e99acc8f9ea8" xlink:to="loc_us-gaap_LicensingAgreementsMember_b349a6d5-38fc-408c-876e-f6d417952995" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsGross_3a016512-01aa-403e-8b6a-be711bbf2b9f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsLineItems_038692ff-6db8-445c-beff-df3c48d259a5" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsGross_3a016512-01aa-403e-8b6a-be711bbf2b9f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization_0938aac6-7d96-45ba-af30-409d9e5d10fb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsLineItems_038692ff-6db8-445c-beff-df3c48d259a5" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization_0938aac6-7d96-45ba-af30-409d9e5d10fb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsNet_c7fd7904-0dcd-43df-b019-e4f4bef53548" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsLineItems_038692ff-6db8-445c-beff-df3c48d259a5" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsNet_c7fd7904-0dcd-43df-b019-e4f4bef53548" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/ShareholdersEquityDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#ShareholdersEquityDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/ShareholdersEquityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfStockByClassTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_us-gaap_StatementClassOfStockAxis_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513" xlink:to="loc_us-gaap_ClassOfStockDomain_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_964d6126-ca9c-450e-b18c-b911de77adec" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_d0af4ecf-dc10-4b9e-8e1c-c0887faf5513" xlink:to="loc_us-gaap_ClassOfStockDomain_964d6126-ca9c-450e-b18c-b911de77adec" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_OrdinarySharesMember_c3ac1f88-a2d6-4ce9-a1b4-a37f05e7f0d7" xlink:href="autl-20250630.xsd#autl_OrdinarySharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_964d6126-ca9c-450e-b18c-b911de77adec" xlink:to="loc_autl_OrdinarySharesMember_c3ac1f88-a2d6-4ce9-a1b4-a37f05e7f0d7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_5ae02201-d82b-42d6-8243-c26e3cc21338" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_5ae02201-d82b-42d6-8243-c26e3cc21338" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_5ae02201-d82b-42d6-8243-c26e3cc21338_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_5ae02201-d82b-42d6-8243-c26e3cc21338" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_5ae02201-d82b-42d6-8243-c26e3cc21338_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_24461700-5588-44fb-b005-330f64df9bfb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_5ae02201-d82b-42d6-8243-c26e3cc21338" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_24461700-5588-44fb-b005-330f64df9bfb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_PublicStockOfferingMember_c55f59ac-e473-41ba-a4ca-e1681a7b7f25" xlink:href="autl-20250630.xsd#autl_PublicStockOfferingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_24461700-5588-44fb-b005-330f64df9bfb" xlink:to="loc_autl_PublicStockOfferingMember_c55f59ac-e473-41ba-a4ca-e1681a7b7f25" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_171d1b42-9aa6-490b-9c6c-970a08502bc4" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_srt_CounterpartyNameAxis_171d1b42-9aa6-490b-9c6c-970a08502bc4" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_171d1b42-9aa6-490b-9c6c-970a08502bc4_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_171d1b42-9aa6-490b-9c6c-970a08502bc4" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_171d1b42-9aa6-490b-9c6c-970a08502bc4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d12e110d-617d-4dfa-8f7c-ce455e454049" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_171d1b42-9aa6-490b-9c6c-970a08502bc4" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d12e110d-617d-4dfa-8f7c-ce455e454049" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_91255df5-f69f-4a4f-aa10-12f0ab6b2db0" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d12e110d-617d-4dfa-8f7c-ce455e454049" xlink:to="loc_autl_BioNTechMember_91255df5-f69f-4a4f-aa10-12f0ab6b2db0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_LegalEntityAxis_81e1289e-3ecb-408d-8828-449dbf21015c" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_LegalEntityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_dei_LegalEntityAxis_81e1289e-3ecb-408d-8828-449dbf21015c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_81e1289e-3ecb-408d-8828-449dbf21015c_default" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_dei_LegalEntityAxis_81e1289e-3ecb-408d-8828-449dbf21015c" xlink:to="loc_dei_EntityDomain_81e1289e-3ecb-408d-8828-449dbf21015c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_498ca67d-15fe-4177-823d-a4810abf9060" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_dei_LegalEntityAxis_81e1289e-3ecb-408d-8828-449dbf21015c" xlink:to="loc_dei_EntityDomain_498ca67d-15fe-4177-823d-a4810abf9060" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_bfdd3756-5fd8-4560-9f54-036ca658a175" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityDomain_498ca67d-15fe-4177-823d-a4810abf9060" xlink:to="loc_autl_BioNTechMember_bfdd3756-5fd8-4560-9f54-036ca658a175" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_99c0f397-7d9f-40da-ab4e-e6befb8c2557" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_us-gaap_AwardTypeAxis_99c0f397-7d9f-40da-ab4e-e6befb8c2557" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_99c0f397-7d9f-40da-ab4e-e6befb8c2557_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_99c0f397-7d9f-40da-ab4e-e6befb8c2557" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_99c0f397-7d9f-40da-ab4e-e6befb8c2557_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_58e75feb-1547-402f-b437-2264b0e3af0e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_99c0f397-7d9f-40da-ab4e-e6befb8c2557" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_58e75feb-1547-402f-b437-2264b0e3af0e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_88a1d7d8-5aff-49fe-90ce-6c894620ee0e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_58e75feb-1547-402f-b437-2264b0e3af0e" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_88a1d7d8-5aff-49fe-90ce-6c894620ee0e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_4a4d1b1f-9489-46e9-99ab-079b325cd175" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_69d97c14-cff9-4d02-9f6e-df388e2b9dfc" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_4a4d1b1f-9489-46e9-99ab-079b325cd175" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_4a4d1b1f-9489-46e9-99ab-079b325cd175_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_4a4d1b1f-9489-46e9-99ab-079b325cd175" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_4a4d1b1f-9489-46e9-99ab-079b325cd175_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_3b59a310-b7da-4117-8a4b-f7ac9e525c73" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_4a4d1b1f-9489-46e9-99ab-079b325cd175" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_3b59a310-b7da-4117-8a4b-f7ac9e525c73" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_b7a04eee-07fb-48b0-87ea-3e617b06003f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_3b59a310-b7da-4117-8a4b-f7ac9e525c73" xlink:to="loc_us-gaap_SubsequentEventMember_b7a04eee-07fb-48b0-87ea-3e617b06003f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CommonStockVotingRightsVotesPerShare_476c6e89-b4c8-451c-900b-79c54d5bbbf3" xlink:href="autl-20250630.xsd#autl_CommonStockVotingRightsVotesPerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_CommonStockVotingRightsVotesPerShare_476c6e89-b4c8-451c-900b-79c54d5bbbf3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsNotIssuedVestedNumber_a0b8fe00-b284-4c56-85b7-2fda7242f7ef" xlink:href="autl-20250630.xsd#autl_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsNotIssuedVestedNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsNotIssuedVestedNumber_a0b8fe00-b284-4c56-85b7-2fda7242f7ef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_4235d12f-0e60-4e50-a107-7883983a22cc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_4235d12f-0e60-4e50-a107-7883983a22cc" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_f4391f46-3cc3-491b-be78-d81bb8edac23" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_f4391f46-3cc3-491b-be78-d81bb8edac23" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockPricePerShare_19baae8e-b859-41b8-b06e-6dd743c2e4a3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockPricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_us-gaap_SaleOfStockPricePerShare_19baae8e-b859-41b8-b06e-6dd743c2e4a3" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_89bbd69e-a641-4027-82e6-234f0d1be854" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_89bbd69e-a641-4027-82e6-234f0d1be854" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_StockholdersEquityCommonStockNumberOfSharesForEachDepositaryShare_85289122-99db-4461-9a43-515e36a21b48" xlink:href="autl-20250630.xsd#autl_StockholdersEquityCommonStockNumberOfSharesForEachDepositaryShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_StockholdersEquityCommonStockNumberOfSharesForEachDepositaryShare_85289122-99db-4461-9a43-515e36a21b48" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CollaborativeArrangementPeriodToEnterAgreementFromInitialClosingDate_31c4651c-a167-42dc-944a-c97af5613f4e" xlink:href="autl-20250630.xsd#autl_CollaborativeArrangementPeriodToEnterAgreementFromInitialClosingDate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_CollaborativeArrangementPeriodToEnterAgreementFromInitialClosingDate_31c4651c-a167-42dc-944a-c97af5613f4e" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementShares_1bc7d829-49fd-401b-902f-caea382545e8" xlink:href="autl-20250630.xsd#autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementShares"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementShares_1bc7d829-49fd-401b-902f-caea382545e8" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementValue_c72ab1eb-0ab9-40d9-892b-9f8f97ab5f43" xlink:href="autl-20250630.xsd#autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_e0d9e229-9bb2-41b1-a9b5-4cfdc36d1224" xlink:to="loc_autl_CollaborativeArrangementRightsAndObligationsMaximumSharePurchaseRequirementValue_c72ab1eb-0ab9-40d9-892b-9f8f97ab5f43" xlink:type="arc" order="9"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/ShareBasedCompensationScheduleofTotalSharebasedCompensationExpenseDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#ShareBasedCompensationScheduleofTotalSharebasedCompensationExpenseDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/ShareBasedCompensationScheduleofTotalSharebasedCompensationExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_838cdf1c-f7bb-4d19-b36f-fc5adb1d6b5c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_6d6417f6-d955-475b-826e-87bb74d71a57" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_838cdf1c-f7bb-4d19-b36f-fc5adb1d6b5c" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_6d6417f6-d955-475b-826e-87bb74d71a57" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationAxis_201ca3a7-5301-4c75-b64f-8cdb87bc9483" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_6d6417f6-d955-475b-826e-87bb74d71a57" xlink:to="loc_us-gaap_IncomeStatementLocationAxis_201ca3a7-5301-4c75-b64f-8cdb87bc9483" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_201ca3a7-5301-4c75-b64f-8cdb87bc9483_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_201ca3a7-5301-4c75-b64f-8cdb87bc9483" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_201ca3a7-5301-4c75-b64f-8cdb87bc9483_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_201ca3a7-5301-4c75-b64f-8cdb87bc9483" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseMember_1904ab2a-8d57-45a4-b4c4-6ffba322ab09" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseMember_1904ab2a-8d57-45a4-b4c4-6ffba322ab09" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingGeneralAndAdministrativeExpensesMember_cea958aa-8931-4bc0-bae6-c8b7c1e28900" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingGeneralAndAdministrativeExpensesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:to="loc_us-gaap_SellingGeneralAndAdministrativeExpensesMember_cea958aa-8931-4bc0-bae6-c8b7c1e28900" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfSalesMember_3e73a49c-3a17-4848-b592-8dac9a12004c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfSalesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:to="loc_us-gaap_CostOfSalesMember_3e73a49c-3a17-4848-b592-8dac9a12004c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CapitalizedIntangibleAssetsNetPropertyAndEquipmentMember_28f45305-fa0d-4fa9-a1b7-ab7c1a40f7c5" xlink:href="autl-20250630.xsd#autl_CapitalizedIntangibleAssetsNetPropertyAndEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_67160409-ff36-4ed4-b1d0-7bf4e3262cd3" xlink:to="loc_autl_CapitalizedIntangibleAssetsNetPropertyAndEquipmentMember_28f45305-fa0d-4fa9-a1b7-ab7c1a40f7c5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_f6913bf4-66b4-40ae-981d-1bb430e8dfa9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_838cdf1c-f7bb-4d19-b36f-fc5adb1d6b5c" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_f6913bf4-66b4-40ae-981d-1bb430e8dfa9" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/ShareBasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#ShareBasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/ShareBasedCompensationNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5ccd4c6a-e932-439e-9bd6-0bc342eb89d4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5ccd4c6a-e932-439e-9bd6-0bc342eb89d4" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_6b4e549a-66e6-40a6-87bd-16b822f680b2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_5ccd4c6a-e932-439e-9bd6-0bc342eb89d4" xlink:to="loc_us-gaap_AwardTypeAxis_6b4e549a-66e6-40a6-87bd-16b822f680b2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_6b4e549a-66e6-40a6-87bd-16b822f680b2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_6b4e549a-66e6-40a6-87bd-16b822f680b2" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_6b4e549a-66e6-40a6-87bd-16b822f680b2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_37c80477-ce86-40b4-8a59-b96de138e9aa" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_6b4e549a-66e6-40a6-87bd-16b822f680b2" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_37c80477-ce86-40b4-8a59-b96de138e9aa" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_71e9951e-f07f-4291-897b-49620bc9b790" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_37c80477-ce86-40b4-8a59-b96de138e9aa" xlink:to="loc_us-gaap_EmployeeStockOptionMember_71e9951e-f07f-4291-897b-49620bc9b790" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_1453f8d1-8b35-47b6-b360-7248ea423367" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_37c80477-ce86-40b4-8a59-b96de138e9aa" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_1453f8d1-8b35-47b6-b360-7248ea423367" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_5cd2088c-4884-49e1-a6c7-735b807c5be6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_5cd2088c-4884-49e1-a6c7-735b807c5be6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue_3a61e770-0e0e-4ec2-8393-c6c104a3d3f5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue_3a61e770-0e0e-4ec2-8393-c6c104a3d3f5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_f76a078f-2941-4e72-ac83-abfceb3da5f7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_f76a078f-2941-4e72-ac83-abfceb3da5f7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_9bd1a7c8-6dae-45c9-afc2-f9f0b6763809" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_9bd1a7c8-6dae-45c9-afc2-f9f0b6763809" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_eeb7369b-f7cb-465e-af63-14ad28d1c74b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_467ba078-1d68-44b5-b3a2-fde5f750e45c" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_eeb7369b-f7cb-465e-af63-14ad28d1c74b" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/ShareBasedCompensationScheduleofRestrictedStockUnitsRSUAwardsActivityDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#ShareBasedCompensationScheduleofRestrictedStockUnitsRSUAwardsActivityDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/ShareBasedCompensationScheduleofRestrictedStockUnitsRSUAwardsActivityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_d63e951c-931c-420f-8beb-eb54c2272465" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_ab006e81-6c79-46e4-868f-4e6ec0bb3442" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_d63e951c-931c-420f-8beb-eb54c2272465" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_ab006e81-6c79-46e4-868f-4e6ec0bb3442" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_ab006e81-6c79-46e4-868f-4e6ec0bb3442" xlink:to="loc_us-gaap_AwardTypeAxis_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_3357436d-cbec-4392-8075-d1f5cf4e11f1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_4ea8e245-a8e0-4d50-91fb-08ea3320a0e9" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_3357436d-cbec-4392-8075-d1f5cf4e11f1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_e8f1e3c1-5933-462b-8b6e-de4d0aacf069" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_3357436d-cbec-4392-8075-d1f5cf4e11f1" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_e8f1e3c1-5933-462b-8b6e-de4d0aacf069" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_feb849c4-9fe5-4f91-b394-a37788b075b1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_d63e951c-931c-420f-8beb-eb54c2272465" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_feb849c4-9fe5-4f91-b394-a37788b075b1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_9d0713c3-14ee-4f25-bd51-bbbc211e5683" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_feb849c4-9fe5-4f91-b394-a37788b075b1" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_9d0713c3-14ee-4f25-bd51-bbbc211e5683" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_b77b3d55-feb5-465c-a17b-9e69511d8c92" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_feb849c4-9fe5-4f91-b394-a37788b075b1" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_b77b3d55-feb5-465c-a17b-9e69511d8c92" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_c5cd8bde-72e6-4a13-97b7-ea7d40e39543" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_feb849c4-9fe5-4f91-b394-a37788b075b1" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_c5cd8bde-72e6-4a13-97b7-ea7d40e39543" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_5b80777b-01fb-451a-80c6-08b4ae770dbd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_bac89f9c-d0a6-4ea4-ad6b-8bd78e5679ae" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_d63e951c-931c-420f-8beb-eb54c2272465" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_bac89f9c-d0a6-4ea4-ad6b-8bd78e5679ae" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_53ba6703-2cb6-4590-99f1-9a4c8787e425" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_bac89f9c-d0a6-4ea4-ad6b-8bd78e5679ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_53ba6703-2cb6-4590-99f1-9a4c8787e425" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_5685380a-60ca-4e1e-a5fc-8e73d5cfeb39" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_bac89f9c-d0a6-4ea4-ad6b-8bd78e5679ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_5685380a-60ca-4e1e-a5fc-8e73d5cfeb39" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_00793efc-dacc-45b8-8d08-f19e1955b9c1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_bac89f9c-d0a6-4ea4-ad6b-8bd78e5679ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_00793efc-dacc-45b8-8d08-f19e1955b9c1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_802835da-c15b-47eb-8c35-41e63b11a626" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/LiabilitiesRelatedtoFutureRoyaltiesandMilestonesNetNarrativeDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#LiabilitiesRelatedtoFutureRoyaltiesandMilestonesNetNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/LiabilitiesRelatedtoFutureRoyaltiesandMilestonesNetNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_96049473-4187-4df1-a4ef-6c04f3f58cf8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_96049473-4187-4df1-a4ef-6c04f3f58cf8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_649ad62a-b27e-4946-a5ea-d41c2e7433b1" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_96049473-4187-4df1-a4ef-6c04f3f58cf8" xlink:to="loc_srt_CounterpartyNameAxis_649ad62a-b27e-4946-a5ea-d41c2e7433b1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_649ad62a-b27e-4946-a5ea-d41c2e7433b1_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_649ad62a-b27e-4946-a5ea-d41c2e7433b1" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_649ad62a-b27e-4946-a5ea-d41c2e7433b1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e79fa27-6443-4e0e-ad8e-cc9572ebf08b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_649ad62a-b27e-4946-a5ea-d41c2e7433b1" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e79fa27-6443-4e0e-ad8e-cc9572ebf08b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BlackstoneMember_966657ca-27e0-4fc8-97c1-0592485dd898" xlink:href="autl-20250630.xsd#autl_BlackstoneMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e79fa27-6443-4e0e-ad8e-cc9572ebf08b" xlink:to="loc_autl_BlackstoneMember_966657ca-27e0-4fc8-97c1-0592485dd898" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_e46b4fda-d622-434d-9450-80956feea786" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e79fa27-6443-4e0e-ad8e-cc9572ebf08b" xlink:to="loc_autl_BioNTechMember_e46b4fda-d622-434d-9450-80956feea786" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_632fd19b-d097-4156-9499-5a5974738940" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_96049473-4187-4df1-a4ef-6c04f3f58cf8" xlink:to="loc_srt_ProductOrServiceAxis_632fd19b-d097-4156-9499-5a5974738940" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_632fd19b-d097-4156-9499-5a5974738940_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_632fd19b-d097-4156-9499-5a5974738940" xlink:to="loc_srt_ProductsAndServicesDomain_632fd19b-d097-4156-9499-5a5974738940_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_0510f651-1a01-4dca-ad24-63317c942b5f" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_632fd19b-d097-4156-9499-5a5974738940" xlink:to="loc_srt_ProductsAndServicesDomain_0510f651-1a01-4dca-ad24-63317c942b5f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ObeCelProductMember_96b69275-b7bb-4913-a2c0-1e48c13474f0" xlink:href="autl-20250630.xsd#autl_ObeCelProductMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_0510f651-1a01-4dca-ad24-63317c942b5f" xlink:to="loc_autl_ObeCelProductMember_96b69275-b7bb-4913-a2c0-1e48c13474f0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpense_9cb74a25-1ea6-4145-88b8-7834f3c8719b" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpense_9cb74a25-1ea6-4145-88b8-7834f3c8719b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityCumulativeCatchUpAdjustment_ec06a7f8-de45-49f1-af28-56071cbd3126" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityCumulativeCatchUpAdjustment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityCumulativeCatchUpAdjustment_ec06a7f8-de45-49f1-af28-56071cbd3126" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityGross_86d6ab22-13c9-4bd0-8e1e-4f80e8caea4b" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityGross_86d6ab22-13c9-4bd0-8e1e-4f80e8caea4b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_NumberOfDevelopmentPayments_65509917-5259-425b-b2d7-d45f18f2f812" xlink:href="autl-20250630.xsd#autl_NumberOfDevelopmentPayments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_NumberOfDevelopmentPayments_65509917-5259-425b-b2d7-d45f18f2f812" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiability_33812a55-1a2b-4665-8adb-4ed5b8965b36" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_RoyaltyAndMilestoneLiability_33812a55-1a2b-4665-8adb-4ed5b8965b36" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount_2b152a7c-e110-4aca-9f03-0c320267534c" xlink:href="autl-20250630.xsd#autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_RevenueRemainingPerformanceObligationVariableConsiderationAmount_2b152a7c-e110-4aca-9f03-0c320267534c" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CollaborativeAgreementInitialRecognitionOfLiability_9e3e5668-e089-4247-b9a2-c3c32e83568c" xlink:href="autl-20250630.xsd#autl_CollaborativeAgreementInitialRecognitionOfLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_autl_CollaborativeAgreementInitialRecognitionOfLiability_9e3e5668-e089-4247-b9a2-c3c32e83568c" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentInterestRateEffectivePercentage_653af464-6d7d-45b8-ae9d-a7368a3d470c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentInterestRateEffectivePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_a4a170d8-b87e-4b1d-b130-821e91c894c9" xlink:to="loc_us-gaap_DebtInstrumentInterestRateEffectivePercentage_653af464-6d7d-45b8-ae9d-a7368a3d470c" xlink:type="arc" order="7"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/LeasesScheduleofMaturitiesofOperatingLeaseLiabilitiesDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#LeasesScheduleofMaturitiesofOperatingLeaseLiabilitiesDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/LeasesScheduleofMaturitiesofOperatingLeaseLiabilitiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LessorLeaseDescriptionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LessorLeaseDescriptionTable_9fb0a93f-e892-4612-a3d6-023b95ea961f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LessorLeaseDescriptionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LessorLeaseDescriptionTable_9fb0a93f-e892-4612-a3d6-023b95ea961f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementScenarioAxis_39a24aa8-f280-4337-b701-c82c49404ebf" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementScenarioAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LessorLeaseDescriptionTable_9fb0a93f-e892-4612-a3d6-023b95ea961f" xlink:to="loc_srt_StatementScenarioAxis_39a24aa8-f280-4337-b701-c82c49404ebf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioUnspecifiedDomain_39a24aa8-f280-4337-b701-c82c49404ebf_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioUnspecifiedDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementScenarioAxis_39a24aa8-f280-4337-b701-c82c49404ebf" xlink:to="loc_srt_ScenarioUnspecifiedDomain_39a24aa8-f280-4337-b701-c82c49404ebf_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioUnspecifiedDomain_51c5f8ca-344a-458b-9c1b-8097012ce3a7" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioUnspecifiedDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementScenarioAxis_39a24aa8-f280-4337-b701-c82c49404ebf" xlink:to="loc_srt_ScenarioUnspecifiedDomain_51c5f8ca-344a-458b-9c1b-8097012ce3a7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioForecastMember_bac0e5ac-de5e-45d6-8cb5-64d10a2dd661" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioForecastMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ScenarioUnspecifiedDomain_51c5f8ca-344a-458b-9c1b-8097012ce3a7" xlink:to="loc_srt_ScenarioForecastMember_bac0e5ac-de5e-45d6-8cb5-64d10a2dd661" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_LesseeOperatingLeaseLiabilityToBePaidRemainderOfFiscalYearNetOfLeaseIncentives_f03bf4c3-0d73-45bf-acda-fc5bb267dc17" xlink:href="autl-20250630.xsd#autl_LesseeOperatingLeaseLiabilityToBePaidRemainderOfFiscalYearNetOfLeaseIncentives"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_autl_LesseeOperatingLeaseLiabilityToBePaidRemainderOfFiscalYearNetOfLeaseIncentives_f03bf4c3-0d73-45bf-acda-fc5bb267dc17" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_LesseeOperatingLeaseLiabilityToBePaidYearOneNetOfLeaseIncentives_16a49ad4-4905-4b58-a5cc-67ff67d51c3e" xlink:href="autl-20250630.xsd#autl_LesseeOperatingLeaseLiabilityToBePaidYearOneNetOfLeaseIncentives"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_autl_LesseeOperatingLeaseLiabilityToBePaidYearOneNetOfLeaseIncentives_16a49ad4-4905-4b58-a5cc-67ff67d51c3e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo_f5aa3fed-6caa-4dd0-bfae-9c9fc8079fc7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo_f5aa3fed-6caa-4dd0-bfae-9c9fc8079fc7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree_6a4782d3-9903-487d-9e0a-87feb6a765c0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree_6a4782d3-9903-487d-9e0a-87feb6a765c0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour_ca51a9d1-2b54-41b2-b039-dc1bfeb7c877" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour_ca51a9d1-2b54-41b2-b039-dc1bfeb7c877" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_LesseeOperatingLeaseLiabilityToBePaidAfterYearFour_23862be1-fca2-4da9-b588-e4d55f2d504c" xlink:href="autl-20250630.xsd#autl_LesseeOperatingLeaseLiabilityToBePaidAfterYearFour"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_autl_LesseeOperatingLeaseLiabilityToBePaidAfterYearFour_23862be1-fca2-4da9-b588-e4d55f2d504c" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue_716b0691-a70f-4188-bf30-1ffe82d18ec6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue_716b0691-a70f-4188-bf30-1ffe82d18ec6" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount_b1c82701-a85f-47b2-99cb-6f4e58c48b20" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount_b1c82701-a85f-47b2-99cb-6f4e58c48b20" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseLiability_8d80de7a-b1f3-4d82-95ab-5fd2df39110d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_OperatingLeaseLiability_8d80de7a-b1f3-4d82-95ab-5fd2df39110d" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncentiveToLessee_45b75c05-1484-4b9d-aebf-b3b0d1e91e4d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncentiveToLessee"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LessorLeaseDescriptionLineItems_e48b629c-d265-445d-895e-e9a77b62c322" xlink:to="loc_us-gaap_IncentiveToLessee_45b75c05-1484-4b9d-aebf-b3b0d1e91e4d" xlink:type="arc" order="9"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/CommitmentsandContingenciesDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#CommitmentsandContingenciesDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/CommitmentsandContingenciesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_6fc4814e-679d-4eb5-801e-7820feaab68b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:to="loc_srt_CounterpartyNameAxis_6fc4814e-679d-4eb5-801e-7820feaab68b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6fc4814e-679d-4eb5-801e-7820feaab68b_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_6fc4814e-679d-4eb5-801e-7820feaab68b" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6fc4814e-679d-4eb5-801e-7820feaab68b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d38c0ac3-3092-48a4-bda0-1971abd6d75e" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_6fc4814e-679d-4eb5-801e-7820feaab68b" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d38c0ac3-3092-48a4-bda0-1971abd6d75e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_UCLBusinessLtdUCLBMember_443d0ff2-2922-4fda-85c5-045eb1c54176" xlink:href="autl-20250630.xsd#autl_UCLBusinessLtdUCLBMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_d38c0ac3-3092-48a4-bda0-1971abd6d75e" xlink:to="loc_autl_UCLBusinessLtdUCLBMember_443d0ff2-2922-4fda-85c5-045eb1c54176" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_9623a5f4-c02f-4f81-9f26-66d091f5cda4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_9623a5f4-c02f-4f81-9f26-66d091f5cda4" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_9623a5f4-c02f-4f81-9f26-66d091f5cda4_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_9623a5f4-c02f-4f81-9f26-66d091f5cda4" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_9623a5f4-c02f-4f81-9f26-66d091f5cda4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_84f028bb-a6fc-47d7-aacf-d8d85dfc9662" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_9623a5f4-c02f-4f81-9f26-66d091f5cda4" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_84f028bb-a6fc-47d7-aacf-d8d85dfc9662" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_7320915c-bbc0-4e75-be81-1c57beb55622" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_84f028bb-a6fc-47d7-aacf-d8d85dfc9662" xlink:to="loc_us-gaap_SubsequentEventMember_7320915c-bbc0-4e75-be81-1c57beb55622" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_92fa5967-c0fa-463b-9b3c-3848111f759f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_92fa5967-c0fa-463b-9b3c-3848111f759f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_92fa5967-c0fa-463b-9b3c-3848111f759f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_92fa5967-c0fa-463b-9b3c-3848111f759f" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_92fa5967-c0fa-463b-9b3c-3848111f759f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_257b1542-f6c9-4f74-8be3-011d6f6f8bc7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_92fa5967-c0fa-463b-9b3c-3848111f759f" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_257b1542-f6c9-4f74-8be3-011d6f6f8bc7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IntellectualPropertyMember_23883d80-55f4-4d9e-973a-c0a4646f0d31" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IntellectualPropertyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_257b1542-f6c9-4f74-8be3-011d6f6f8bc7" xlink:to="loc_us-gaap_IntellectualPropertyMember_23883d80-55f4-4d9e-973a-c0a4646f0d31" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_190613a8-bce4-4648-91b5-8a601de17b68" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_2a057ebb-fd7a-4623-bd68-21777d77fc77" xlink:to="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_190613a8-bce4-4648-91b5-8a601de17b68" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_190613a8-bce4-4648-91b5-8a601de17b68_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_190613a8-bce4-4648-91b5-8a601de17b68" xlink:to="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_190613a8-bce4-4648-91b5-8a601de17b68_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_fda62146-a758-4a71-8321-408e3f947c3b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationByCategoryOfItemPurchasedAxis_190613a8-bce4-4648-91b5-8a601de17b68" xlink:to="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_fda62146-a758-4a71-8321-408e3f947c3b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalAdditionsMember_1f4b2a66-1bec-4a64-a03c-a8cd5be3e5e9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalAdditionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_fda62146-a758-4a71-8321-408e3f947c3b" xlink:to="loc_us-gaap_CapitalAdditionsMember_1f4b2a66-1bec-4a64-a03c-a8cd5be3e5e9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_MasterSupplyCommitmentsMember_b8f87b4a-94c6-491f-ace0-96d6b30ef720" xlink:href="autl-20250630.xsd#autl_MasterSupplyCommitmentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_UnconditionalPurchaseObligationCategoryOfGoodsOrServicesAcquiredDomain_fda62146-a758-4a71-8321-408e3f947c3b" xlink:to="loc_autl_MasterSupplyCommitmentsMember_b8f87b4a-94c6-491f-ace0-96d6b30ef720" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpense_9073b05b-7df4-4c52-bc5c-741bf42d6763" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpense_9073b05b-7df4-4c52-bc5c-741bf42d6763" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsToAcquireIntangibleAssets_b5be80ba-eaef-4a29-bb66-1b214a172668" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsToAcquireIntangibleAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_us-gaap_PaymentsToAcquireIntangibleAssets_b5be80ba-eaef-4a29-bb66-1b214a172668" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount_69f9b307-1408-4559-b3cf-ec006ee53997" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_us-gaap_UnrecordedUnconditionalPurchaseObligationBalanceSheetAmount_69f9b307-1408-4559-b3cf-ec006ee53997" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_DistributionAgreementTerm_f4214cf6-f943-4fdf-acbd-3db148cadabc" xlink:href="autl-20250630.xsd#autl_DistributionAgreementTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_autl_DistributionAgreementTerm_f4214cf6-f943-4fdf-acbd-3db148cadabc" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualAtCarryingValue_5a44587d-bf8d-43a8-bae3-dbcaae7c3011" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingencyAccrualAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_us-gaap_LossContingencyAccrualAtCarryingValue_5a44587d-bf8d-43a8-bae3-dbcaae7c3011" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtained_eebec7b7-24ba-4040-8042-44e37cbc93b4" xlink:href="autl-20250630.xsd#autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtained"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtained_eebec7b7-24ba-4040-8042-44e37cbc93b4" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtainedNetBenefit_21e12025-5567-49b2-ad51-6c2e8017f91b" xlink:href="autl-20250630.xsd#autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtainedNetBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_6298d8fe-f9e4-4c2f-b2c7-7920bfb33761" xlink:to="loc_autl_SMEBenefitThresholdReductionInTheValueOfTheTaxCreditObtainedNetBenefit_21e12025-5567-49b2-ad51-6c2e8017f91b" xlink:type="arc" order="6"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/SegmentReportingScheduleofLongLivedAssetsbyGeographicLocationDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#SegmentReportingScheduleofLongLivedAssetsbyGeographicLocationDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/SegmentReportingScheduleofLongLivedAssetsbyGeographicLocationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_eac96a51-c094-4221-bfc3-73b81d52bd04" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_2678ed41-eda8-4422-8996-b6586897b95d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_eac96a51-c094-4221-bfc3-73b81d52bd04" xlink:to="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_2678ed41-eda8-4422-8996-b6586897b95d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_bca79adc-da11-4732-a8df-be3ff1184b9b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable_2678ed41-eda8-4422-8996-b6586897b95d" xlink:to="loc_srt_StatementGeographicalAxis_bca79adc-da11-4732-a8df-be3ff1184b9b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_bca79adc-da11-4732-a8df-be3ff1184b9b_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_bca79adc-da11-4732-a8df-be3ff1184b9b" xlink:to="loc_srt_SegmentGeographicalDomain_bca79adc-da11-4732-a8df-be3ff1184b9b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_6376354c-0cb4-4a53-8cce-656a43f47b4a" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_bca79adc-da11-4732-a8df-be3ff1184b9b" xlink:to="loc_srt_SegmentGeographicalDomain_6376354c-0cb4-4a53-8cce-656a43f47b4a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_GB_4d7112f5-4e4f-4640-9388-c5f2c62640de" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_GB"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_6376354c-0cb4-4a53-8cce-656a43f47b4a" xlink:to="loc_country_GB_4d7112f5-4e4f-4640-9388-c5f2c62640de" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_US_7253dcb4-b894-4458-8bd2-d9aa8172ed95" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_6376354c-0cb4-4a53-8cce-656a43f47b4a" xlink:to="loc_country_US_7253dcb4-b894-4458-8bd2-d9aa8172ed95" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NoncurrentAssets_f53dcf7d-16be-45c2-8b2e-62acb118b6e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NoncurrentAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems_eac96a51-c094-4221-bfc3-73b81d52bd04" xlink:to="loc_us-gaap_NoncurrentAssets_f53dcf7d-16be-45c2-8b2e-62acb118b6e7" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/SegmentReportingScheduleofSegmentLossIncludingSignificantSegmentExpensesDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#SegmentReportingScheduleofSegmentLossIncludingSignificantSegmentExpensesDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/SegmentReportingScheduleofSegmentLossIncludingSignificantSegmentExpensesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_294bea93-94f5-4dc6-b0cc-c196623d547d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_294bea93-94f5-4dc6-b0cc-c196623d547d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_5d6e0807-0123-4fed-b98c-4b128b42daf6" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_294bea93-94f5-4dc6-b0cc-c196623d547d" xlink:to="loc_srt_ProductOrServiceAxis_5d6e0807-0123-4fed-b98c-4b128b42daf6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_5d6e0807-0123-4fed-b98c-4b128b42daf6_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_5d6e0807-0123-4fed-b98c-4b128b42daf6" xlink:to="loc_srt_ProductsAndServicesDomain_5d6e0807-0123-4fed-b98c-4b128b42daf6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_ae98bfe4-7704-44aa-99fa-cc0130bde88b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_5d6e0807-0123-4fed-b98c-4b128b42daf6" xlink:to="loc_srt_ProductsAndServicesDomain_ae98bfe4-7704-44aa-99fa-cc0130bde88b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProductMember_1407146a-0c41-41d3-ba93-c5a60cef5877" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProductMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_ae98bfe4-7704-44aa-99fa-cc0130bde88b" xlink:to="loc_us-gaap_ProductMember_1407146a-0c41-41d3-ba93-c5a60cef5877" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicenseMember_bb4ee768-3851-4f9d-8e08-04edf72e2135" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LicenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_ae98bfe4-7704-44aa-99fa-cc0130bde88b" xlink:to="loc_us-gaap_LicenseMember_bb4ee768-3851-4f9d-8e08-04edf72e2135" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_294bea93-94f5-4dc6-b0cc-c196623d547d" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0" xlink:to="loc_us-gaap_SegmentDomain_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_d35ca67f-f32c-4613-badd-fcffbadc2b58" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_3b7fd32d-fefb-4ee7-afcb-203dd4b177c0" xlink:to="loc_us-gaap_SegmentDomain_d35ca67f-f32c-4613-badd-fcffbadc2b58" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ReportableSegmentMember_a7b5146b-5c2b-4c28-b7be-7dbd6dcd9a1c" xlink:href="autl-20250630.xsd#autl_ReportableSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_d35ca67f-f32c-4613-badd-fcffbadc2b58" xlink:to="loc_autl_ReportableSegmentMember_a7b5146b-5c2b-4c28-b7be-7dbd6dcd9a1c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_2f73a9e4-07fa-4fb0-b03d-f7bd650f05e1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_2f73a9e4-07fa-4fb0-b03d-f7bd650f05e1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpensesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfGoodsAndServicesSold_268618e7-fa4b-46ed-b9f9-f34957850eda" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfGoodsAndServicesSold"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_us-gaap_CostOfGoodsAndServicesSold_268618e7-fa4b-46ed-b9f9-f34957850eda" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ResearchAndClinicalDevelopmentExpenses_08800df4-b670-4a53-89bb-f5bfed5dc66b" xlink:href="autl-20250630.xsd#autl_ResearchAndClinicalDevelopmentExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_autl_ResearchAndClinicalDevelopmentExpenses_08800df4-b670-4a53-89bb-f5bfed5dc66b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_ProductDeliveryExpense_1e2ccf90-3fc7-421b-b8f3-1d53037c9d00" xlink:href="autl-20250630.xsd#autl_ProductDeliveryExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_autl_ProductDeliveryExpense_1e2ccf90-3fc7-421b-b8f3-1d53037c9d00" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_CommercialAndMedicalExpense_d8bca06b-4b80-4529-addc-ab64b334c838" xlink:href="autl-20250630.xsd#autl_CommercialAndMedicalExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_autl_CommercialAndMedicalExpense_d8bca06b-4b80-4529-addc-ab64b334c838" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_SupportFunctionsCosts_e0d0da82-9366-4669-9c59-ac6b8ffaad69" xlink:href="autl-20250630.xsd#autl_SupportFunctionsCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_autl_SupportFunctionsCosts_e0d0da82-9366-4669-9c59-ac6b8ffaad69" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingOtherItemAmount_80f8953e-4f6e-4263-9f0d-4ebcda23bda7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingOtherItemAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_us-gaap_SegmentReportingOtherItemAmount_80f8953e-4f6e-4263-9f0d-4ebcda23bda7" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpenses_0549465b-eda9-4723-9a91-7b3974a2ef8d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_76a2e4a8-3832-48a5-9b9f-0d75e7b16b55" xlink:to="loc_us-gaap_OperatingExpenses_0549465b-eda9-4723-9a91-7b3974a2ef8d" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_699984ba-45f4-4dcd-b322-648d341fbeb3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_OperatingIncomeLoss_699984ba-45f4-4dcd-b322-648d341fbeb3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherNonoperatingIncomeExpense_99e2e63d-c736-4bcf-87fb-51efaf91309f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherNonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_OtherNonoperatingIncomeExpense_99e2e63d-c736-4bcf-87fb-51efaf91309f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignCurrencyTransactionGainLossBeforeTax_5fa60e2c-2654-4538-9811-efd7feace9d9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCurrencyTransactionGainLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_ForeignCurrencyTransactionGainLossBeforeTax_5fa60e2c-2654-4538-9811-efd7feace9d9" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentIncomeInterest_f24f64ac-feea-4918-9691-b1369d2375ca" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InvestmentIncomeInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_InvestmentIncomeInterest_f24f64ac-feea-4918-9691-b1369d2375ca" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestExpenseNonoperating_eb0c5cb3-9b0f-4c1b-9b2f-87ea52a963f3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterestExpenseNonoperating"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_InterestExpenseNonoperating_eb0c5cb3-9b0f-4c1b-9b2f-87ea52a963f3" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_a10c3107-237c-4121-8926-66e5a575d457" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_a10c3107-237c-4121-8926-66e5a575d457" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_24ee40f3-b7c4-4356-9216-2983b8a220d9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_45e3b09d-4ba1-4a01-872b-8d82109ef2cb" xlink:to="loc_us-gaap_NetIncomeLoss_24ee40f3-b7c4-4356-9216-2983b8a220d9" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.autolus.com/role/RelatedPartyTransactionsDetails" xlink:type="simple" xlink:href="autl-20250630.xsd#RelatedPartyTransactionsDetails"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/RelatedPartyTransactionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RelatedPartyTransactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_9e985201-5f95-4526-b998-f789b1eb7e08" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_9e985201-5f95-4526-b998-f789b1eb7e08" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_6e11d966-92ed-40b0-9bc9-2fd091254e92" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_9e985201-5f95-4526-b998-f789b1eb7e08" xlink:to="loc_srt_CounterpartyNameAxis_6e11d966-92ed-40b0-9bc9-2fd091254e92" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e11d966-92ed-40b0-9bc9-2fd091254e92_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_6e11d966-92ed-40b0-9bc9-2fd091254e92" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_6e11d966-92ed-40b0-9bc9-2fd091254e92_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_c4a45982-4833-4571-8690-677b94a4be12" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_6e11d966-92ed-40b0-9bc9-2fd091254e92" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_c4a45982-4833-4571-8690-677b94a4be12" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BlackstoneMember_9fc3be0f-37f2-4b17-8e37-dda23c2f94c5" xlink:href="autl-20250630.xsd#autl_BlackstoneMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_c4a45982-4833-4571-8690-677b94a4be12" xlink:to="loc_autl_BlackstoneMember_9fc3be0f-37f2-4b17-8e37-dda23c2f94c5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_BioNTechMember_4fc22f0a-225e-4f28-9d3c-b42f46f21a34" xlink:href="autl-20250630.xsd#autl_BioNTechMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_c4a45982-4833-4571-8690-677b94a4be12" xlink:to="loc_autl_BioNTechMember_4fc22f0a-225e-4f28-9d3c-b42f46f21a34" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_OwnershipAxis_b6194b67-1170-448f-ab72-cfd6b9f3ce31" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_OwnershipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_9e985201-5f95-4526-b998-f789b1eb7e08" xlink:to="loc_srt_OwnershipAxis_b6194b67-1170-448f-ab72-cfd6b9f3ce31" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_OwnershipDomain_b6194b67-1170-448f-ab72-cfd6b9f3ce31_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_OwnershipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_OwnershipAxis_b6194b67-1170-448f-ab72-cfd6b9f3ce31" xlink:to="loc_srt_OwnershipDomain_b6194b67-1170-448f-ab72-cfd6b9f3ce31_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_OwnershipDomain_9319bc55-cbcd-424f-a6d8-2e07759e8262" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_OwnershipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_OwnershipAxis_b6194b67-1170-448f-ab72-cfd6b9f3ce31" xlink:to="loc_srt_OwnershipDomain_9319bc55-cbcd-424f-a6d8-2e07759e8262" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_AutolusMember_5ec705b5-61b3-4d71-a4ec-7a9d65ad8469" xlink:href="autl-20250630.xsd#autl_AutolusMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_OwnershipDomain_9319bc55-cbcd-424f-a6d8-2e07759e8262" xlink:to="loc_autl_AutolusMember_5ec705b5-61b3-4d71-a4ec-7a9d65ad8469" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MinorityInterestOwnershipPercentageByNoncontrollingOwners_4f06690f-c2b7-47bf-ad25-3064816b0126" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MinorityInterestOwnershipPercentageByNoncontrollingOwners"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_us-gaap_MinorityInterestOwnershipPercentageByNoncontrollingOwners_4f06690f-c2b7-47bf-ad25-3064816b0126" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_NumberOfDirectors_ec43c0a9-a68d-4463-ac98-689c724b0a37" xlink:href="autl-20250630.xsd#autl_NumberOfDirectors"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_autl_NumberOfDirectors_ec43c0a9-a68d-4463-ac98-689c724b0a37" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiability_cda8030e-005f-4685-a84a-cdd009b683e3" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_autl_RoyaltyAndMilestoneLiability_cda8030e-005f-4685-a84a-cdd009b683e3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpenseAndCumulativeCatchUpAdjustment_40659a17-5a39-4123-bb34-805e007a3bdc" xlink:href="autl-20250630.xsd#autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpenseAndCumulativeCatchUpAdjustment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_autl_RoyaltyAndMilestoneLiabilityNonCashInterestExpenseAndCumulativeCatchUpAdjustment_40659a17-5a39-4123-bb34-805e007a3bdc" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_autl_PaymentsForRoyaltiesAndMilestones_3705ef69-dfe3-4625-b2eb-9736defef8e0" xlink:href="autl-20250630.xsd#autl_PaymentsForRoyaltiesAndMilestones"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_018e45bc-852f-45f1-9046-ee84b730f543" xlink:to="loc_autl_PaymentsForRoyaltiesAndMilestones_3705ef69-dfe3-4625-b2eb-9736defef8e0" xlink:type="arc" order="4"/>
  </link:definitionLink>
</link:linkbase>
