Skeletal myogenesis is a well-studied differentiation process. However, despite the identification and functional characterization of the myogenic basic HLH transcription factors, molecular details are still lacking. With the advent of microarray technology, it has become possible to look at changes in gene expression profiles in a biological process on an unprecedented scale. In this study, we applied this technology to profile gene expression during the in vitro differentiation of an established myoblast cell line, C2C12. We report over 1500 genes whose expression is altered when these cells differentiate, including 624, or about 40% of the total number of genes, with unknown functions. This analysis reveals the existence of 12 groups of coordinately regulated genes that are expressed in temporal waves of gene expression prior to the transcriptional induction of myogenin. Among these are multiple families of transcription factors that are important for the process of myogenesis. In addition, the induction of the Notch signaling pathway suggests that previously unappreciated intercellular signaling occurs during myogenic differentiation. These results provide a molecular description of the skeletal myogenesis up to the activation of myogenin.