The transplantation of cultured myoblasts into mature skeletal muscle is a promising therapeutic approach for muscle and non-muscle diseases, known as myoblast-mediated gene therapy. The success of this therapy depends on the fusion of implanted cells with host myofibers. Previous studies using established myogenic cell lines or primary muscle cultures have faced challenges due to the tumorigenic nature of cell lines and the presence of non-myogenic cells in primary cultures. To overcome these issues, the authors developed novel culture conditions that allow the purification of mouse myoblasts from primary cultures. These enriched and clonal populations of primary myoblasts were characterized for cell proliferation and differentiation. Primary myoblasts required bFGF for growth and retained their differentiation potential even after 30 population doublings. The fate of these cells after transplantation was monitored using β-galactosidase (β-gal) labeling, which revealed that primary myoblasts fused with host muscle cells to form hybrid myofibers within five days of transplantation. In syngeneic hosts, these hybrid fibers persisted for up to six months without immune suppression, while in allogeneic hosts, the transplanted cells were rapidly rejected. Additionally, primary myoblasts showed no tumorigenicity, unlike C2 myoblasts, which formed tumors in immunodeficient mice. The ease of isolation, growth, and transfection of primary mouse myoblasts under these conditions expands the opportunities for studying muscle cell growth and differentiation, both in normal and mutant strains of mice, and suggests that primary myoblasts can be broadly applied to mouse models of human muscle and non-muscle diseases.The transplantation of cultured myoblasts into mature skeletal muscle is a promising therapeutic approach for muscle and non-muscle diseases, known as myoblast-mediated gene therapy. The success of this therapy depends on the fusion of implanted cells with host myofibers. Previous studies using established myogenic cell lines or primary muscle cultures have faced challenges due to the tumorigenic nature of cell lines and the presence of non-myogenic cells in primary cultures. To overcome these issues, the authors developed novel culture conditions that allow the purification of mouse myoblasts from primary cultures. These enriched and clonal populations of primary myoblasts were characterized for cell proliferation and differentiation. Primary myoblasts required bFGF for growth and retained their differentiation potential even after 30 population doublings. The fate of these cells after transplantation was monitored using β-galactosidase (β-gal) labeling, which revealed that primary myoblasts fused with host muscle cells to form hybrid myofibers within five days of transplantation. In syngeneic hosts, these hybrid fibers persisted for up to six months without immune suppression, while in allogeneic hosts, the transplanted cells were rapidly rejected. Additionally, primary myoblasts showed no tumorigenicity, unlike C2 myoblasts, which formed tumors in immunodeficient mice. The ease of isolation, growth, and transfection of primary mouse myoblasts under these conditions expands the opportunities for studying muscle cell growth and differentiation, both in normal and mutant strains of mice, and suggests that primary myoblasts can be broadly applied to mouse models of human muscle and non-muscle diseases.