It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression-independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11(+) leukemia samples. Interestingly, Cbfb-MYH11(+) preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were Csf2rb(+). Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression-independent activities contribute to leukemogenesis by Cbfb-MYH11.