The generation of B lymphocyte precursors is dependent on the combinatorial action of the transcription factors PU.1, Ikaros, E2A, EBF, and Pax-5. Loss of PU.1 results in a severe reduction in Flk2⁺, IL-7R⁺ lymphoid progenitors as well as impaired expression of EBF and Pax-5. Restoration of EBF expression facilitates rapid generation of pro-B cells from PU.1^−/− progenitors. Molecular analysis suggests that PU.1 directly participates in regulation of the EBF gene. Although PU.1 is dispensable for expression of most early B lineage genes, it is required for CD45R/B220. Using EBF^−/− mutant progenitors, we show that EBF induces Pax-5 and the early program of B lineage gene expression. Importantly, Pax-5 does not rescue B cell development from either PU.1^−/− or EBF^−/− progenitors. Pax-5 expression and function are contingent on EBF. Based on these results, we propose a hierarchical regulatory network for specification and commitment to the B cell fate.