Oligodendrocytes, the myelinating cells in the vertebrate central nervous system, electrically insulate axons and facilitate rapid propagation of action potentials. The isolation and transplantation of oligodendroglial precursor cells (OPCs) could provide a powerful means to characterize their differentiation properties and potential and enhance myelin repair. Preparation and maintenance of rodent OPCs have been a challenge due to difficulties in obtaining a sufficient quantity. Here, we describe step-by-step and efficient methods to prepare highly enriched OPCs from rat and mice. Isolated OPCs can be induced to differentiate into oligodendrocytes and express most, if not all, of oligodendrocyte-associated genes in vivo. The ability to isolate and culture OPCs should facilitate studies on oligodendrocyte lineage progression and their utility in myelin repair after injury.