The generation of an oligodendrocyte primary culture model encompassing the diverse stages of the lineage is essential for the in vitro research of oligodendrocyte physiology and pathophysiology. Here, we provide a protocol for generating oligodendrocytes from the neonatal rodent brain. We describe steps for isolating oligodendrocyte progenitor cells (OPCs) through differential centrifugation, their subsequent expansion, passaging, and differentiation. For complete details on the use and execution of this protocol, please refer to Kim et al.1.