In this study, we have investigated the relationship of Olig2+ and Nkx2.2+ oligodendrocyte progenitors (OLPs) by comparing the expression of Olig2 and Nkx2.2 in embryonic chicken and mouse spinal cords before and during the stages of oligodendrogenesis. At the stages of neurogenesis, Olig2 and Nkx2.2 are expressed in adjacent non-overlapping domains of ventral neuroepithelium. During oligodendrogenesis stages, these two domains generate distinct populations of OLPs. From the Olig2+ motoneuron precursor domain (pMN) arise the Olig2+/Pdgfra+ OLPs, whereas the Nkx2.2+ p3 domain give rise to Nkx2.2+ OLPs. Despite their distinct origins, both populations of OLPs eventually appear to co-express Olig2 and Nkx2.2 in the same cells. However, there is a species difference in the timing of acquiring Nkx2.2 expression by the Olig2+/Pdgfra+ OLPs. The co-expression of Nkx2.2 and Olig2 in OLPs is tightly associated with myelin gene expression in the normal and PDGFA(-/-) embryos, suggesting a cooperative role of these transcription factors in the control of oligodendrocyte differentiation. In support of this suggestion, inhibition of expression of these two transcription factors in culture by antisense oligonucleotides has an additive inhibitory effect on OLP differentiation and proteolipid protein (PLP) gene expression.