Core-binding factor β (CBFβ) and CBFα2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFβ or CBFα2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFβ and CBFα2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion ofCBFB with MYH11, the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for aCbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb−/−embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfβ in hematopoietic cells. In midgestation embryos, Cbfβ was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and thatCbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfβ was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfβ was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.