MyoD is sufficient to initiate the skeletal muscle gene expression program. Transcription of certain MyoD-target genes occurs in the early phases while that of others is induced only at later stages of differentiation, even though MyoD is present throughout the differentiation process. MyoD acetylation regulates transcriptional competency, yet whether this post-translational modification is equally relevant for activation of all the MyoD targets is unknown. Moreover, the molecular mechanisms through which acetylation ensures that MyoD achieves its optimal activity remain unexplored. To address these two outstanding issues, we have coupled genome-wide expression profiling and chromatin immunoprecipitation in a model system in which MyoD or its non-acetylatable version was inducibly activated in mouse embryonic fibroblasts derived from MyoD-/-/Myf5-/- mice. Our results reveal that MyoD acetylation influences transcription of selected genes expressed at defined stages of the muscle program by regulating chromatin access of MyoD, histone acetylation, and RNA polymerase II recruitment.