In CBFβ-SMMHC, core binding factor beta (CBFβ) is fused to the α-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFβ-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFβ-SMMHC(ΔACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the ΔACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G₁ to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFβ domain to 149 or 83 C-terminal CBFβ-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFβ-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFβ-SMMHC(83C) did not. The majority of CBFβ-SMMHC and CBFβ-SMMHC(149C) was detected in the nucleus, whereas the ΔACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFβ-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFβ-SMMHC(ΔACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFβ-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G₁ to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFβ-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFβ-SMMHC expression.