Dysregulation of paracrine signaling in oligodendrocyte progenitor cells (OPCs) and impaired oligodendrocyte differentiation likely contribute to the failure of remyelination in human demyelinating diseases such as multiple sclerosis (MS). Following demyelination of myelinated axons, OPCs are recruited to lesioned areas wherein they differentiate into myelinating oligodendrocytes capable of repleting myelin to damaged axons. In MS, OPC differentiation, and thus remyelination, is hindered, leading to impartial repair and chronic neurological deficits. Recent studies have shown that muscarinic M1/3 receptor (M1/3R) activation inhibits OPC differentiation, and pharmacological or genetic blockade of M1/3R leads to enhanced OPC differentiation and remyelination in a number of animal and human models. However, the pathological role of endogenous muscarinic agonist, acetylcholine (ACh) ligand-dependent receptor signaling in OPCs versus constitutive receptor activation remains unclear. To test the ligand dependent signaling hypothesis, we utilize mice demyelination models (lysoelcithin and cuprizone) and viral mediated ChAT ablation strategies to elucidate the role of cholinergic signaling on OPC differentiation and remyelination.