Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation ofCd79a(mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences ofEbf1haploinsufficiency together with haploinsufficiency ofRunx1genes in mice. Although numbers of “committed” pro-B cells were maintained inEbf1+/−Runx1+/−(ERhet) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, κ0sterile transcripts, CD2 and CD25 were reduced and delayed inERhetpro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells inERhetmice. Late pre-B and immature and mature B cells were decreased in the bone marrow ofEbf1+/−(Ehet) mice and were nearly absent inERhetmice. Although we did not observe significant effects of haploinsuficiencies onIgHorIgκ rearrangements, a relative lack ofIgλ rearrangements was detected inEhetandERhetpre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow ofEhetandERhetmice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reducedEbf1dosage and the compounding effects of reducedRunx1dosage. Our data confirm and extend the importance of EBF1 in regulating target genes andIggene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.