The bHLH (basic helix-loop-helix) transcription factor Mash1 is best known for its role in the regulation of neurogenesis. However, Mash1 is also expressed in oligodendrocyte precursors and has recently been shown to promote the generation of oligodendrocytes in cell culture, suggesting that it may regulate oligodendrogenesis as well. Here, we show that in the developing ventral forebrain, Mash1 is expressed by a subset of oligodendrocyte precursors (OPCs) as soon as they are generated in the ventricular zone. Using reporter mice, we demonstrate that a subset of OPCs in both the embryonic and postnatal forebrain originate from Mash1-positive progenitors, including a large fraction of adult NG2-positive OPCs. Using Mash1 null mutant mice, we show that Mash1 is required for the generation of an early population of OPCs in the ventral forebrain between embryonic day 11.5 (E11.5) and E13.5, whereas OPCs generated later in embryonic development are not affected. Overexpression of Mash1 in the dorsal telencephalon induces expression of PDGFRalpha (platelet-derived growth factor receptor alpha) but not other OPC markers, suggesting that Mash1 specifies oligodendrogenesis in cooperation with other factors. Analysis of double-mutant mice suggests that Olig2 is one of the factors that cooperate with Mash1 for generation of OPCs. Together, our results show for the first time that Mash1 cooperates in vivo with Olig2 in oligodendrocyte specification, demonstrating an essential role for Mash1 in the generation of a subset of oligodendrocytes and revealing a genetic heterogeneity of oligodendrocyte lineages in the mouse forebrain.