Satellite cells are myogenic stem cells responsible for the postnatal regeneration of skeletal muscle. Here we report the successful in vitro induction of Pax7‐positive satellite‐like cells from mouse embryonic stem (mES) cells. Embryoid bodies were generated from mES cells and cultured on Matrigel‐coated dishes with Dulbecco's modified Eagle medium containing fetal bovine serum and horse serum. Pax7‐positive satellite‐like cells were enriched by fluorescence‐activated cell sorting using a novel anti‐satellite cell antibody, SM/C‐2.6. SM/C‐2.6‐positive cells efficiently differentiate into skeletal muscle fibers both in vitro and in vivo. Furthermore, the cells demonstrate satellite cell characteristics such as extensive self‐renewal capacity in subsequent muscle injury model, long‐term engraft‐ment up to 24 wk, and the ability to be secondarily transplanted with remarkably high engraftment efficiency compared to myoblast transplantation. This is the first report of transplantable, functional satellite‐like cells derived from mES cells and will provide a foundation for new therapies for degenerative muscle disor‐ders.—Chang, H.,Yoshimoto, M., Umeda, K., Iwasa, T., Mizuno, Y., Fukada, S., Yamamoto, H., Motohashi, N., Yuko‐Miyagoe‐Suzuki, Takeda, S., Heike, T., Nakahata, T. Generation of transplantable, functional satellite‐like cells from mouse embryonic stem cells. FASEB J. 23, 1907–1919 (2009)