The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor β (CBFβ) also binds to the RD to enhance RD–DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFβ and in a ternary complex with DNA and CBFβ. Consistent with the experimental findings, in the presence of CBFβ the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the ‘wing’ residues (RD residues 139–145) and the ‘wing1’ residues (RD residues 104–116). The simulation studies also indicate that the RD–CBFβ binding is more favorable in the presence of DNA due to a more favorable RD–CBFβ interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD–CBFβ interaction is predicted.