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Here, we used high-dimensional flow cytometry, transcriptomic analysis, functional assays, and precursor-product experiments to define the development pathway of V\u03b39V\u03b42 T cells in the postnatal thymus. We identify three distinct stages of development for V\u03b39V\u03b42 T cells in the postnatal thymus that are defined by the progressive acquisition of functional potential and major changes in the expression of transcription factors, chemokines, and other surface markers. Furthermore, our analysis of donor-matched thymus and blood revealed that the molecular requirements for the development of functional V\u03b39V\u03b42 T cells are delivered predominantly by the postnatal thymus and not in the periphery. Tbet and Eomes, which are required for IFN-\u03b3 and TNF\u03b1 expression, are up-regulated as V\u03b39V\u03b42 T cells mature in the thymus, and mature thymic V\u03b39V\u03b42 T cells rapidly express high levels of these cytokines after stimulation. Similarly, the postnatal thymus programs V\u03b39V\u03b42 T cells to express the cytolytic molecules, perforin, granzyme A, and granzyme K.\u00a0This study provides a greater understanding of how V\u03b39V\u03b42 T cells develop in humans and may lead to opportunities to manipulate these cells to treat human diseases.<\/jats:p>","DOI":"10.1126\/sciimmunol.abo4365","type":"journal-article","created":{"date-parts":[[2023,7,14]],"date-time":"2023-07-14T17:58:37Z","timestamp":1689357517000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":44,"title":["A three-stage developmental pathway for human V\u03b39V\u03b42 T cells within the postnatal thymus"],"prefix":"10.1126","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-7949-8597","authenticated-orcid":true,"given":"Louis","family":"Perriman","sequence":"first","affiliation":[{"name":"Murdoch Children\u2019s Research Institute, Melbourne, Australia."},{"name":"Department 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