The differentiation of the bipotential O2A progenitor cell into an oligodendrocyte or a type 2 astrocyte has been well documented in cell cultures of various regions of the central nervous system. The appropriate tools to prove its existence in vivo have been lacking. We report on an in vitro-in vivo approach that combines stable labeling of an enriched population of cultured O2A progenitors by the fluorescent dye fast blue, followed by their transplantation into neonatal rat brains, which allowed us to study the influence of the brain microenvironment on their lineage decision. The grafted cells survived well and 21 days after grafting nearly all were positive for the oligodendroglial marker galactocerebroside. Surprisingly, the fast blue-positive grafted cells did not stain for the astroglial marker glial fibrillary acidic protein. These results indicate that the O2A progenitor's plasticity is restricted by the in vivo environment, resulting in the developmental exclusion of the type 2 astrocyte initially described in vitro.