Precise control of hematopoietic stem cell (HSC) proliferation and differentiation is needed to maintain a lifetime supply of blood cells. Using genome-wide ENU mutagenesis and phenotypic screening, we have identified a mouse line that harbors a point mutation in the transactivation (TA) domain of the transcription factor c-Myb (M303V), which reduces c-Myb-dependent TA by disrupting its interaction with the transcriptional coactivator p300. The biological consequences of the c-Myb^M303V/M303V mutation include thrombocytosis, megakaryocytosis, anemia, lymphopenia, and the absence of eosinophils. Detailed analysis of hematopoiesis in c-Myb^M303V/M303V mice reveals distinct blocks in T cell, B cell, and red blood cell development, as well as a remarkable 10-fold increase in the number of HSCs. Cell cycle analyses show that twice as many HSCs from c-Myb^M303V/M303V animals are actively cycling. Thus c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells.