Regenerative medicine is expected to be a potent therapeutic option for the disorders or injuries of the central nervous system. However, little is known about how the newly formed neurons derived from grafted stem cells integrate into the host established tissue. The aims of this study are to make functional connection between primary neurons and stem cell-derived neurons via chemical synapses and maintain the connection for a long time in in vitro. We employed an in vitro co-culture device to cultivate two different neuronal populations and evaluate the interaction between them. Mouse cortical neurons and P19 cell-derived neurons were co-cultured in the co-culture device. The synchronous activities were maintained for at least 4 weeks. Evoked responses to electrical stimulation suggested that bi-directional connections were formed between cortical and P19-derived neurons. The responses were changed after pharmacological treatment. These results showed that cortical neurons and P19 cell-derived neurons formed bidirectional synaptic connections via glutamate receptors and the connection was maintained for at least 4 weeks.