Objective: To investigate in vitro differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes in chemical conditional medium. Methods: The mixed glial cells from cerebral cortices of 48-hour-old Sprague-Dawley (SD) rats were cultured in vitro. The OPCs were separated by shaking procedure around 9–10 d in the primary culture. Then the isolated OPCs were further transferred into the chemical conditional medium for cell differentiation. The pattern of OPCs maturation in vitro was continuously observed with contrast phase microscopy and mature oligodendrocytes were further identified by immunocytochemical assays. Results: OPCs grew well when co-cultured with glial cells and distinct cellular stratification formed about 9–10 d in the primary culture, which indicated the appropriate opportunity for the separation of OPCs. Following cultured in the chemical conditional medium, the OPCs progressively differentiated into the mature oligodendrocytes. These mature oligodendrocytes were also immunostained with the oligodendrocyte lineage-specific antibody, Oligo2. Conclusion: The OPCs isolated from the cerebral cortices of neonatal SD rats can progressively differentiate into mature oligodendrocytes in the chemical conditional medium in vitro. Objective: To investigate in vitro differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes in chemical conditional medium. Methods: The mixed glial cells from cerebral cortices of 48-hour-old Sprague-Dawley (SD) rats were cultured in vitro. OPCs were separated by shaking procedure around 9-10 d in the primary culture. Then the isolated OPCs were further transferred into the chemical conditional medium for cell differentiation. The pattern of OPCs maturation in vitro was continuously observed with contrast phase microscopy and mature oligodendrocytes were further identified by immunocytochemical assays. Results: OPCs grew well when co-cultured with glial cells and distinct cellular stratification formed about 9-10 d in the primary culture, which indicates the appropriate opportunity for the separation of OPCs. Following cultured in the chemical conditional medium, the OPCs progressively differentiated into the mature oligodendrocytes oligodendrocytes were also immunostained with the oligodendrocyte lineage-specific antibody, Oligo 2. Conclusion: The OPCs isolated from the cerebral cortices of neonatal SD rats can be progressively differentiate into mature oligodendrocytes in the chemical conditional medium in vitro.