Oligodendrocytes are derived from glial precursors that arise from the ventral neural tube early in development. In the developing chicken CNS, oligodendrocyte progenitors selectively express Nkx2.2 homeodomain transcription factor, raising the possibility that Nkx2.2 may directly regulate oligogliogenesis. In this study, we have examined Nkx2.2 expression in rodent glial precursors and studied the effect of a loss of Nkx2.2 on oligodendrocyte and astrocyte differentiation. We show that Nkx2.2 is also expressed in mammalian oligodendrocyte progenitors and that the differentiation of MBP-positive and PLP-DM20-positive oligodendrocytes is dramatically retarded in Nkx2.2-null mutants along the entire rostrocaudal axis. In contrast, no effect is seen on astrocytic differentiation. Interestingly, absence of Nkx2.2 expression leads to a ventral expansion of the Olig1/Olig2 expression in neuroepithelial cells into the Nkx2.2 domain and a consequent increase in the production of Olig1/Olig2-positive and platelet-derived growth factor receptor alpha-positive oligodendrocyte progenitors. These results strongly suggest that Nkx2.2 regulates the differentiation and/or maturation, but not the initial specification, of oligodendrocyte progenitors. Consistent with this suggestion, overproduction of Nkx2.2 protein in fibroblast cells can induce gene expression from the proteolipid protein promoter.