The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.