We have previously shown that oligodendroglial progenitors (OP) can be generated from multipotent rat neural precursor cells. We now report the generation of a homogeneous culture of canine OP from neural precursor cells. In non-adherent cultures, homogeneous OP cultures were obtained in 6-8 weeks of treatment with B104 cell conditioned medium (B104CM). In adherent cultures where astrocytes grew as a layer of substrate, colonies of OP invariably appeared at 10-14 days in vitro (DIV) and the colonies were expanded as free-floating spheres (oligospheres), in the presence of B104CM, suggesting that astrocytes facilitate the generation of canine OP. The oligosphere cells were characterized by self-renewal in the presence of B104CM and by terminal differentiation into oligodendrocytes after withdrawal of B104CM. Transplantation studies indicated that the extensively expanded oligosphere cells retained myelination capacity. The oligospheres thus provide a valuable source for experimental cell therapy studies.