Oligodendrocyte progenitor cells (OPCs, NG2 glia) play an important role not only as progenitor cells that give rise to myelinating cells in the central nervous system (CNS), but also as an active participant in the neural network. It is necessary to develop a simplified method for generating large quantities of highly purified OPCs for biochemical studies and to establish a neuron-OPC coculture method for functional studies on the mechanism of neuron-OPC signaling. In this study, we have compared the effects of plating density and culture medium on purity, survival, and differentiation of cells collected from primary rat mixed glial cultures by differential adhesion. Comparison of two chemically-defined culture media, Dulbecco's modified Eagle's medium with N1 supplements (N1/DMEM) and Neurobasal medium with B27 supplements (B27/NBM) revealed that while both media successfully maintained greater than 90% pure OPCs after 3 days, B27/NBM was significantly more effective in maintaining viable cells and in supporting oligodendrocyte differentiation than N1/DMEM, and this effect was more pronounced in low density cultures. Furthermore, B27/NBM supported neuron-OPC coculture in which OPCs remained as NG2-positive progenitors and neurons differentiated to form synapses over a period of 3 weeks.