CBFbeta-SMMHC is expressed in 8% of acute myeloid leukemias and inhibits AML1/RUNX1. In this study, murine marrow or human CD34(+) cells were transduced with retroviral or lentiviral vectors expressing CBFbeta-SMMHC or two mutant variants. CBFbeta-SMMHC reduced murine or human myeloid cell proliferation three- to four-fold in liquid culture relative to empty vector-transduced cells, during a period when vector-transduced cells accumulated five-fold and human cells 20-fold. CBFbeta-SMMHC decreased the formation of myeloid, but not erythroid, colonies two- to four-fold, and myeloid colonies expressing CBFbeta-SMMHC were markedly reduced in size. However, CBFbeta-SMMHC did not slow differentiation to granulocytes or monocytes. Neither CBFbeta-SMMHC(Delta2-11), which does not bind AML1, nor CBFbeta-SMMHC(DeltaACD), which does not multimerize or efficiently bind corepressors, slowed proliferation or reduced myeloid colonies. CBFbeta-SMMHC increased the G1/S ratio 1.4-fold. AML1 had an effect opposite to CBFbeta-SMMHC, stimulating proliferation of murine myeloid progenitors 2.0-fold in liquid culture. Thus, CBFbeta-SMMHC directly inhibits the proliferation of normal myeloid progenitors via inhibition of AML1 and dependent upon the integrity of its assembly competence domain. These findings support the development of therapeutics that target the ability of CBFbeta-SMMHC to interact with AML1 or to multimerize via its assembly competence domain.