NSCs are found in the developing brain, as well as in the adult brain. They are self-renewing cells that maintain the capacity to differentiate into all major brain-specific cell types, such as glial cells and neurons. However, it is still unclear whether these cells are capable of gaining full functionality, which is one of the major prerequisites for NSC-based cell replacement strategies of neurological diseases. The ability to establish and maintain polarized excitatory synaptic contacts would be one of the basic requirements for intercellular communication and functional integration into existing neuronal networks. In primary cultures of hippocampal neurons, it has already been shown that synaptogenesis is characterized by a well-ordered, time-dependent targeting and recruitment of pre- and postsynaptic proteins. In this study, we investigated the expression and localization of important pre- and postsynaptic proteins, including Bassoon and synaptophysin, as well as proteins of the ProSAP/Shank family, in differentiating rat fetal mesencephalic NSCs. Moreover, we analyzed the ultrastructural features of neuronal cell-cell contacts during synaptogenesis. We show that NSCs express and localize cytoskeletal and scaffolding molecules of the pre- and postsynaptic specializations in a well-defined temporal order, leading to mature synaptic contacts after 14 days of differentiation. The temporal and spatial pattern of synaptic maturation is comparable to synaptogenesis of hippocampal neurons grown in primary culture. Therefore, with respect to the general ability to create mature synaptic contacts, NSCs seem to be well equipped to potentially compensate for lost or injured brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.