The transcription factors EBF and E2A are required at a similar step in early B cell differentiation. EBF and E2A synergistically upregulate transcription of endogenous B cell-specific genes in a non-B cell line. Here, we examine a genetic collaboration between these factors in regulating B lymphopoiesis. We find that Ebf+/- E2a+/- mice display a marked defect in pro-B cell differentiation at a stage later than observed in the single homozygous mutant mice. Pro-B cells from Ebf+/- E2a+/- mice show reduced expression of lymphoid-specific transcripts, including Pax5, Rag1, Rag2, and mb-1. We also show that EBF directly binds and activates the Pax5 promoter. Together, these data show collaboration between EBF and E2A and provide insight into the hierarchy of transcription factors that regulate B lymphocyte differentiation.