The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFβ causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease, expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis, little is known about the contribution of CBFβ-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFβ-MYH11 cDNA into human CD34⁺ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture, resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B₂M^-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFβ-SMMHC on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology, and in defining its contribution to the development of leukemia.