The B lineage commitment factor Pax5 (BSAP) is exclusively expressed in B lymphocytes of the blood system. To study the effect of Pax5 on the development of other hematopoietic lineages, we generated a heterozygous knockin mouse carrying a Pax5 minigene under the control of the Ikaros locus. Conditional and constitutive activation of the Ik(Pax5) allele demonstrated that precocious Pax5 expression in hematopoietic stem cells and progenitors failed to interfere with myeloid development and only weakly affected erythroblast formation. Instead, pan-hematopoietic Pax5 expression strongly promoted B cell development at the expense of T lymphopoiesis. Pax5 thereby interfered with T lineage commitment and early thymocyte development by repressing the transcription of the T cell specification gene Notch1.