We have established a novel, simple, and highly reproducible method to generate skeletal muscle cells from mouse skin. Small pieces of skin from the back of mice were cultured in extracellular material-coated dishes in typical culture medium for about 3 weeks. Myotubes formed after about a week, grew into twitching myotubes, and became twitching myotube clumps after 3 weeks. Skeletal muscle cells are formed spontaneously with no induction. Myotubes were immunologically positive for myosin heavy chains, MyoD, and myogenin. Ultrastructural analysis revealed the presence of the sarcomere structure. Furthermore, PAX7(+)/MyoD(-) muscle stem cells proliferated around these myotubes, and MyoD(+)/myogenin(+)/MHC- cells were also observed. Moreover, we investigated the formation of skeletal muscle cells from the sialidosis mouse skin, and showed that it is decreased compared to that of the wild type. Our method to generate skeletal muscle cells from skin is thought to be useful for the investigation of muscle cell development and muscle-related disorders. (C) 2020 Elsevier Inc. All rights reserved.