Using in vitro systems, the proliferation, migration, differentiation, and survival of immature oligodendrocyte lineage cells can be examined to elucidate the cellular and molecular interactions that regulate this lineage. The ability to monitor progressive stages of differentiation within the lineage by immunophenotyping and to manipulate the cellular responses with growth factors makes these cultures advantageous as both a method for studying the cell biology of myelination and as a model system for lineage analysis in the mammalian central nervous system. In addition, cultured oligodendrocytes carry out the normal in vivo sequence of expression of a set of cell type-specific genes, some of which are extremely highly expressed, and so provide advantages for analysis of gene regulation. This paper describes commonly used methods for the preparation of mixed glial cell cultures from perinatal rodent brain. Although these cultures are most commonly derived from perinatal rat brain, a protocol for preparation from mouse brain is also provided because of the increasing number of studies that use mice to facilitate molecular biological techniques. Methods to prepare secondary cultures of different stages of oligodendrocyte lineage cells are detailed. As examples of methods to use for the characterization of these cells, immunophenotypes of each stage of the oligodendrocyte lineage are illustrated, incorporation of [3H]thymidine for analysis of cell proliferation is illustrated, and detailed methods are provided for analysis of migration in a microchemotaxis chamber.