Human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells (MSCs) are invariably present in UCB, several reports showed that MSC-like cells could be consistently derived from human UCB and, moreover, could differentiate into various cells of a mesodermal origin. However, it remains unclear whether these UCB-derived MSCs are also capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion, UCB-derived mononuclear cells gave rise to adherent layers of fibroblast-like cells expressing MSC-related antigens such as SH2, SH3, alpha-smooth muscle actin, CD13, CD29, and CD49e. More important, when these UCB-derived MSCs were incubated in promyogenic conditions for up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation pattern. Both flow cytometric and reverse transcriptase-polymerase reaction analyses showed that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of incubation but not after 2 weeks. At week 6, more than half of UCB-derived MSCs expressed myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB-derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle-related tissue engineering.