Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBF alpha subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBF beta subunit. CBF alpha binds DNA in a sequence-specific manner, whereas CBF beta enhances DNA binding by CBF alpha. Both DNA binding and heterodimerization with CBF beta are mediated by a single domain in the CBF alpha subunits known as the "Runt domain." We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBF beta. We identified two energetic "hot spots" that were also found in a similar analysis of CBF beta (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579-39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBF beta interface.