The decision to develop into αβ or γδ T cells is pre-programmed in distinct subpopulations of DN1 thymocytes

T cells are divided into the αβ and γδ lineages. It is currently thought that these lineages differentiate in the thymus from uncommitted progenitor thymocytes. However, in this study we show that the decision to differentiate into one or either lineage is in fact already fixed in these apparent uncommitted progenitors. Using single-cell RNA sequencing, we reassemble de novo a model of early T cell development based on the transcriptional profiles of individual CD4-CD8- double negative and γδ thymocytes. We show that the earliest thymocyte stage, known as CD4-CD8- double negative 1 (DN1), is actually comprised of a mixture of transcriptionally distinct subpopulations. Although not yet expressing definitive markers of αβ and γδ lineages, such as the lineage-defining T cell receptors, specific DN subpopulations exhibit restricted developmental potential for either αβ or γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. Thus, T cell lineage decisions are hardwired from the earliest stages of T cell development. ### Competing Interest Statement The authors have declared no competing interest.