With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.