The basic helix-loop-helix (bHLH) transcription factor Ascl1 plays crucial roles in both oligodendrocyte development and neuronal development; however, the molecular target of Ascl1 in oligodendrocyte progenitor cells (OPCs) remains elusive. To identify the downstream targets of Ascl1 in OPCs, we performed gene expression microarray analysis and identified Hes5 as a putative downstream target of Ascl1. In vivo analysis revealed that Ascl1 and Hes5 were coexpressed in early developmental oligodendrocytes in both the telencephalon and the ventral spinal cord. We also found that Hes5 expression was reduced in the OPCs of Ascl1 mutant mice. Furthermore, we demonstrated that Ascl1 directly binds to an E-box region within the Hes5 promoter and regulates Hes5 expression at the transcriptional level. Taken together, these in vivo and in vitro data suggest that Ascl1 induces Hes5 expression in a cell-autonomous manner. Considering the previously known function of Hes5 as a repressor of Ascl1, our data indicate that Hes5 is involved in the negative feedback regulation of Ascl1. © 2012 Wiley Periodicals, Inc.