Objective To obtain healthier and purer oligodendrocyte precursor cells (OPCs) via modification of the methods in OPCs isolation, culture and generation passage, and to mimic the development of OPCs in vivo via setup of an in vitro model of lineage restricted oligodendrocyte differentiation. Methods Using nonspecific differentiated pre-attachment and A2B5IgM immuno-panning, OPCs isolated from embryonic rat spinal cords were highly purified. Their proliferations were induced by enrichment of the culture solution with fibroblast growth factor-2 and platelet-derived growth factor, and their differentiations to mature oligodendrocytes were induced with tri-iodothyronine and neurotrophin-3. The morphology and characteristics of OPCs and cells differentiated from them were observed using microscope and immunostaining. Results More than 95% of the cells exhibited typical OPCs morphology of bipolar or tripolar processes, and expressed A2B5. These cells could proliferate steadily and execute generation passage repeatedly. After inducement into differentiation process, cells expressed specific antigens, such as O4, O1, receptor-interacting protein and myelin basic protein, sequentially. Conclusion The morphology, proliferation and differentiation of the isolated OPCs are similar with the O2A precursor cells in embryonic brain (natural OPCs).