Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBFβ), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBFβ fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBFβ-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBFα) with higher affinity than the native CBFβ protein. NMR studies identify interactions in the CBFβ portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.