Oligodendrocytes (OLs) are glial cells of the central nervous system, which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs) as well as providing a model to study human OL development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during OL differentiation including neural progenitor cells, glial progenitor cells and oligodendrocyte progenitor cells (OPCs) compared to undifferentiated ESCs. Using a 1% false discovery rate cutoff, ∼3145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as transferrin, neural cell adhesion molecule 1, apolipoprotein E and wingless-related MMTV integration site 5A showed increased expression from the neural progenitor cell to the OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including fatty acid-binding protein 4, THBS1, bone morphogenetic protein 1, CRYAB, transferrin, tenascin C, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human ESC differentiation into OPCs, this study provides many novel proteins that are potentially involved in OL development.