Background: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic moto-neuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. Methods: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. Results: This unique serum-and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. Conclusion: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.