The DNA methylation inhibitor 5-azacytidine is widely used to stimulate the cardiac differentiation of stem cells. However, 5-azacytidine has long been employed as a tool for stimulating skeletal myogenesis. Yet, it is unclear whether the ability of 5-azacytidine to promote both cardiac and skeletal myogenesis is dependent strictly on the native potential of the starting cell population or if this drug is a transdifferentiation agent. To address this issue, we examined the effect of 5-azacytidine on cultures of adult mouse atrial tissue, which contains cardiac but not skeletal muscle progenitors. Exposure to 5-azacytidine caused atrial cells to elongate and increased the presence of fat globules within the cultures. 5-Azacytidine also induced expression of the skeletal myogenic transcription factors MyoD and myogenin. 5-Azacytidine pretreatments allowed atrial cells to undergo adipogenesis or skeletal myogenesis when subsequently cultured with either insulin and dexamethasone or low-serum media, respectively. The presence of skeletal myocytes in atrial cultures was indicated by dual staining for myogenin and sarcomeric α-actin. These data demonstrate that 5-azacytidine converts cardiac cells to noncardiac cell types and suggests that this drug has a compromised efficacy as a cardiac differentiation factor.