In the development of embryo, motor neurons from spinal cords extend their axons to skeletal muscle cells to form neuromuscular junctions and muscle contractions are achieved thereafter. Although skeletal muscles cells secrete factors that affect survival and growth of motor neurons, much has remained unclear. Embryonic stem (ES) cells differentiate into motor neurons when they are treated with retinoic acid and sonic hedgehog in vitro, and C2C12 cells are well-known mouse myoblasts. Therefore, our aims are to clarify the interactions between motor neurons from mouse ES cells and skeletal muscle cells from C2C12 in vitro. While previous studies have used co-culture systems in which two cell types are maintained at the same time in a chamber, this system does not mimic embryonic development in vivo and it is difficult to analyze the interactions precisely. We then, developed new co-culture system using dual chambers in which motor neurons derived from mouse ES cells and C2C12 myoblasts were incubated independently. After a certain time period, walls of the dual chambers were removed and two types of cells were finally co-cultured. Immuno-fluorescence, RT-PCR and K252a were used to analyze the interactions between motor neurons and skeletal muscle cells. As a result, it was found that the number of survival motor neurons increased and the length of the extending dendrites and axons was prolonged. RT-PCR dates showed mRNA expressions of neurotrophic factors in skeletal muscle cells and their receptors in motor neurons, respectively. The mRNA expressions in co-culture groups were different from those of single culture groups. Furthermore, the number of surviving motor neurons and the length of neurites decreased by K252a treatments. The results suggest that neurotrophic factors derived from skeletal muscles cells promoted survival of motor neurons, and extended neurites via their receptors in motor neurons. Moreover, co-culture of motor neurons and skeletal muscle cells yielded the interaction between them, and then altered the expression of factors and receptors .