The principle function of oligodendrocytes is to synthesize and maintain myelin, a lipid-based substance essential for rapid conduction between neurons. Demyelinating diseases such as multiple sclerosis (MS) are characterized by a loss of oligodendrocytes and their myelin. Remyelination restores myelin but is limited due to impaired human oligodendrocyte progenitor cell (hOPC) differentiation. Unfortunately, current understanding of the genes involved in human oligodendrocyte development is lacking. To expand our knowledge in this field, we sought to identify and characterize genes that regulate differentiation by extracting mRNA from CD140a+ hOPCs undergoing oligodendrocyte differentiation in vitro. Microarray-based differential gene expression analysis identified 49 genes that were greatly regulated in OPC differentiation. Weighted Gene Co-expression Network Analysis (WGCNA) was used to define differentiation-specific networks and identified GNB4, SULF2, PPP161RB and ITM2A as genes that may act as key regulators of hOPC differentiation. Interestingly, lentiviral over-expression of GNB4 and SULF2 could significantly promote and inhibit OPC differentiation respectively in vitro. We sought to validate the functions of these genes in vivo. As such, we transplanted lentiviral infected hOPCs over-expressing SULF2, GNB4, or mCherry, the control, into the corpus callosum of neonatal shiverer/rag2 hypomyelinating mice. At 8 and 12 weeks post-implantation we perfused, sectioned, and stained their brains via immunohistochemistry using markers for proliferation (Ki67), and differentiation as astrocyte (GFAP) and oligodendrocyte lineages (CC1 and MBP). At 8 weeks, GNB4-infected hOPC transplanted animals exhibited more than a 3 fold increase in MBP within the corpus callosum (unpaired t-test p < 0.05, n=4). SULF2 over-expression greatly reduced the percentage of transplanted cells undergoing CC1+ oligodendrocyte differentiation (9.8 ± 0.6% vs mCherry 15.9 ± 2%, unpaired t-test p = 0.013, n=4). These results confirm the roles of GNB4 and SULF2 in hOPC differentiation and suggest that SULF2 and GNB4 could represent future pharmacological targets for remyelinating therapy.