Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.