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

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2024 Workiva-->
<!--r:41d517a8-76a2-4778-a70e-daa514c34351,g:458e9ce8-ec2b-4777-be06-f5aade039560-->
<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/Cover" xlink:type="simple" xlink:href="autl-20240517.xsd#Cover"/>
  <link:definitionLink xlink:role="http://www.autolus.com/role/Cover" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityListingsLineItems_cd2b39da-f8bb-4bfe-87e5-5d03d7119485" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_EntityListingsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityListingsTable_5124931b-34b0-470c-a3aa-f451174d0e6d" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_EntityListingsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_dei_EntityListingsLineItems_cd2b39da-f8bb-4bfe-87e5-5d03d7119485" xlink:to="loc_dei_EntityListingsTable_5124931b-34b0-470c-a3aa-f451174d0e6d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_f950a345-cb60-4b4f-ad6a-261c36868a73" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_dei_EntityListingsTable_5124931b-34b0-470c-a3aa-f451174d0e6d" xlink:to="loc_us-gaap_StatementClassOfStockAxis_f950a345-cb60-4b4f-ad6a-261c36868a73" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_f950a345-cb60-4b4f-ad6a-261c36868a73_default" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_f950a345-cb60-4b4f-ad6a-261c36868a73" xlink:to="loc_us-gaap_ClassOfStockDomain_f950a345-cb60-4b4f-ad6a-261c36868a73_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_971ee391-0c8c-42a0-b05e-7212073675a6" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_f950a345-cb60-4b4f-ad6a-261c36868a73" xlink:to="loc_us-gaap_ClassOfStockDomain_971ee391-0c8c-42a0-b05e-7212073675a6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonClassAMember_cdb1312b-f233-49a3-99c6-813acfff2dc6" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonClassAMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_971ee391-0c8c-42a0-b05e-7212073675a6" xlink:to="loc_us-gaap_CommonClassAMember_cdb1312b-f233-49a3-99c6-813acfff2dc6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_sic_Z8880_b47bc63c-6341-4f0a-bc9e-84480fab4f59" xlink:href="https://xbrl.sec.gov/sic/2022/sic-2022.xsd#sic_Z8880"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_971ee391-0c8c-42a0-b05e-7212073675a6" xlink:to="loc_sic_Z8880_b47bc63c-6341-4f0a-bc9e-84480fab4f59" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_Security12bTitle_b1932c16-a0cd-4912-a278-306e5f941f98" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_Security12bTitle"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityListingsLineItems_cd2b39da-f8bb-4bfe-87e5-5d03d7119485" xlink:to="loc_dei_Security12bTitle_b1932c16-a0cd-4912-a278-306e5f941f98" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_TradingSymbol_0e913ba3-9d00-4be1-bcb9-bfb94e0985ee" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_TradingSymbol"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityListingsLineItems_cd2b39da-f8bb-4bfe-87e5-5d03d7119485" xlink:to="loc_dei_TradingSymbol_0e913ba3-9d00-4be1-bcb9-bfb94e0985ee" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_SecurityExchangeName_8cb5693c-ae26-4779-ac76-1531cae5cc2a" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_SecurityExchangeName"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityListingsLineItems_cd2b39da-f8bb-4bfe-87e5-5d03d7119485" xlink:to="loc_dei_SecurityExchangeName_8cb5693c-ae26-4779-ac76-1531cae5cc2a" xlink:type="arc" order="2"/>
  </link:definitionLink>
</link:linkbase>
