Myelination is the culmination of a complex process in which oligodendrocyte (OL) progenitors transition through defined stages in a well–coordinated differentiation program. The signaling mechanisms that regulate this progression are poorly understood. Here we investigate the role of extracellular signal–regulated–kinase–1,–2 (Erk1/2) and the mammalian target of rapamycin (mTOR), downstream effectors of the Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways, at specific stages of OL development in vitro. Using a panel of developmental stage–specific antigenic markers and pharmacological inhibitors, we provide evidence that Erk1/2 signaling regulates transition of early progenitors to the late progenitor stage and, as a consequence, to the immature OL stage, but not the transition of immature OL to the mature OL stage. In contrast, mTOR signaling is not required for early progenitor transition to late progenitor stage. Surprisingly, it is also not required for the transition of late progenitors to terminally differentiated immature OLs, as has been reported previously, but is required for the next sequential transition of immature OLs to the mature OL stage. Furthermore, mTOR signaling regulates OL cytoskeletal organization and major myelin protein expression. These in vitro findings correlate with our in vivo data showing that inhibition of mTOR by rapamycin injection attenuated the onset of myelination in the early postnatal brain. Thus, these studies demonstrate that Erk1/2 and mTOR signaling sequentially regulates distinct stages of OL progenitor differentiation and suggest that cells in the OL–lineage require distinct signaling mechanisms to transition through specific stages of their development. © 2011 Wiley Periodicals, Inc.