In this report, we identify myogenin as an important transcriptional target under the control of three intracellular signaling pathways, namely, the p38 mitogen-activated protein kinase- (MAPK), calcium-calmodulin-dependent protein kinase- (CaMK), and calcineurin-mediated pathways, during skeletal muscle differentiation. Three cis-elements (i.e., the E box, myocyte enhancer factor [MEF] 2, and MEF3 sites) in the proximal myogenin promoter in response to these three pathways are defined. MyoD, MEF2s, and Six proteins, the trans-activators bound to these cis-elements, are shown to be activated by these signaling pathways. Our data support a model in which all three signaling pathways act in parallel but nonredundantly to control myogenin expression. Inhibition of any one pathway will result in abolished or reduced myogenin expression and subsequent phenotypic differentiation. In addition, we demonstrate that CaMK and calcineurin fail to activate MEF2s in Rhabdomyosarcoma-derived RD cells. For CaMK, we show its activation in response to differentiation signals and its effect on the cytoplasmic translocation of histone deacetylases 5 are not compromised in RD cells, suggesting histone deacetylases 5 cytoplasmic translocation is necessary but not sufficient, and additional signal is required in conjunction with CaMK to activate MEF2 proteins.