The major diversification of Vγ1.1+ and Vγ2+ thymocytes in mice occurs after commitment to the γδ T‐cell lineage

γδ T cells are a heterogeneous cell population with different subsets playing specialized and often opposing roles during immune responses. A key question is whether γδ thymocytes are determined for their effector function already at an early stage, before their commitment to the γδ T‐cell lineage, or are instructed during their later development. Here, we show that the adult Vγ1.1+ and Vγ2+ γδ T‐cell subsets both go through a CD73+CD24+ development stage, and that the gene regulation involved in lineage commitment is shared by both subsets. We demonstrate that the major subset diversification first occurs after the cells have committed to the γδ T‐cell lineage, strongly supporting an instructive model for functional programming of γδ T cells. In conclusion, we show that the two major adult γδ T‐cell subsets in mice develop through a shared pathway utilizing similar cellular machinery and that they diverge after the CD24+CD73+ maturity stage. Immature uncommitted and committed γδ thymocytes are enriched for Vγ2+ and Vγ1.1+ cells, respectively; but exhibit similar stage‐specific global gene expression. Conversely, mature Vγ1.1+ and Vγ2+ thymocytes have drastically diverged, suggesting that the major subset diversification occurs after commitment to the γδ T‐cell lineage. .