Abstract Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5−/− pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5−/− progenitors efficiently differentiate in vitro into CD4+CD8+ αβ and γδ T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5−/− progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRβ, γ, and δ loci in the correct temporal sequence. Notch-stimulated Pax5−/− progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same time, Notch signaling rapidly induces cell surface expression of the c-Kit receptor and transcription of the target genes Deltex1 and pre-Tα concomitant with the activation of TCR Vβ germline transcription and the regulatory genes GATA3 and Tcf1. These data suggest that Notch1 acts upstream of GATA3 and Tcf1 in early T cell development and regulates Vβ-DJβ rearrangements by controlling the chromatin accessibility of Vβ genes at the TCRβ locus.