Recent studies have suggested that oligodendrocyte development is likely to be under the control of a hierarchy of lineage-specific transcription factors. In the developing mouse spinal cord, expression of Olig2, Sox10 and Nkx2.2 is sequentially up-regulated in cells of oligodendrocyte lineage. These transcription factors play essential roles in oligodendrocyte specification and differentiation. However, the regulatory relationship and functional interactions among these transcription factors have not been determined. In this study, we systematically investigated the function and hierarchical relationship of Olig2, Sox10 and Nkx2.2 transcription factors in the control of oligodendrocyte differentiation. It was found that over-expression of Olig2 is sufficient to induce Sox10, Nkx2.2 and precocious oligodendrocyte differentiation in embryonic chicken spinal cord. Sox10 expression alone is also sufficient to stimulate ectopic oligodendrocyte differentiation and weakly induce Nkx2.2 expression. Although genetic evidence indicated that Sox10 functions downstream of Olig2, Sox10 activity can modulate Olig2 expression. In addition, we presented evidence that the control of oligodendrocyte differentiation by Olig2, Sox10 and Nkx2.2 is a dosage-dependent developmental process and can be affected by both haploinsufficiency and over-dosage.