T cell development from expanded hematopoietic progenitors reveals initiation control by and Flt3L priming.

To access the earliest stages of murine T cell development, we adapted a serum-free expansion system for hematopoietic stem and progenitor-like cells. These cells undergo normal T cell differentiation in vivo and in vitro, verified by gene expression trajectories from single-cell RNA sequencing. Their absolute differentiation speed is slower than that of fresh progenitors but is modulated with Flt3L priming. Leveraging this system, the earliest T cell lineage-initiating events in response to Notch signaling included chromatin opening and transcriptional activation of the TCR-Cβ locus. Acute CRISPR knockouts revealed that some transcription factors normally inherited from bone marrow progenitors impede early T cell development, with differing effects on proliferation. Among 23 factors tested, knockout greatly accelerated the onset of germline TCR-Cβ locus transcription and expression of , , and and their targets. Thus, normal endogenous expression of this progenitor- and leukemia-associated factor markedly restrains T cell program initiation.