Skeletal muscle differentiation is controlled by interactions between myocyte enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription factors. Association of MEF2 with histone deacetylases (HDAC) -4 and -5 results in repression of MEF2 target genes and inhibition of myogenesis. Calcium/calmodulin-dependent protein kinase (CaMK) signaling promotes myogenesis by disrupting MEF2-HDAC complexes and stimulating HDAC nuclear export. To further define the mechanisms that confer CaMK responsiveness to HDAC4 and -5, we performed yeast two-hybrid screens to identify HDAC-interacting factors. These screens revealed interactions between HDAC4 and members of the 14-3-3 family of proteins, which function as signal-dependent intracellular chaperones. HDAC4 binds constitutively to 14-3-3 in yeast and mammalian cells, whereas HDAC5 binding to 14-3-3 is largely dependent on CaMK signaling. CaMK phosphorylates serines -259 and -498 in HDAC5, which subsequently serve as docking sites for 14-3-3. Our studies suggest that 14-3-3 binding to HDAC5 is required for CaMK-dependent disruption of MEF2-HDAC complexes and nuclear export of HDAC5, and implicate 14-3-3 as a signal-dependent regulator of muscle cell differentiation.