In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.