Oligodendrocytes are destroyed in demyelinating diseases such as Multiple Sclerosis. In a genomic study to identify novel drug targets, we identified the muscarinic cholinergic M3 receptor as highly expressed by human oligodendrocyte progenitor cells (OPCs). Human OPCs expressing CD140a antigen were isolated from 19–22 week fetal brain using immunomagnetic sorting. We treated OPCs with oxotremorine, a specific agonist of muscarinic receptors, for 4 days and assessed cell fate and proliferation using immunocytochemistry. Oxotremorine treatment resulted in a dose‐dependent decrease in oligodendrocyte differentiation (1‐way ANOVA, p<0.01). The proportion of O4+ oligodendrocytes was significantly reduced from 16.0±2.4% at 0μM to 8.3±1.5% at 40μM oxotremorine (n=3, Tukey's post‐hoc p<0.05). The proportions of dividing cells (BrdU) and oligodendrocyte lineage cells (Olig2) were not significantly altered suggesting that the effect of oxotremorine was to directly block oligodendrocyte differentiation. As such, we have identified an operative receptor expressed by human OPCs that may be targeted to regulate oligodendroctye differentiation. This work was supported by NYSTEM contracts (C026413 and C026428).