Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFα subunit and a non‐DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFα:β:DNA complex. Dissociation constants for all four equilibria involving the CBFα Runt domain, CBFβ, and DNA were defined. Conformational changes associated with interactions between CBFα, CBFβ, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFβ ‘locks in’ a high affinity DNA binding conformation of the CBFα Runt domain.