<p>Multiple sclerosis (MS) is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte (OLG) loss, and progressive neurological decline. While current disease-modifying therapies primarily target immune responses, they offer limited neuroprotective or remyelinating effects. Through its receptors, including cannabinoid receptor type 1 (CB₁R) and the G protein-coupled receptor GPR55, the endocannabinoid system (ECS) has emerged as a promising therapeutic target due to its roles in neuronal survival and glial function. To better study the functional role of these receptors and their potential as targets for remyelinating therapies, we sought a human cell model that lacks endogenous expression of CB₁R and GPR55. Quantitative PCR confirmed that human OLG-derived HOG cells do not express these receptors, providing a clean background for transfection-based functional studies. Using this system, we found that expressing either CB₁R or GPR55 was sufficient to confer resistance to cuprizone (CPZ)-induced cytotoxicity, while co-expression produced a comparable level of protection but uncovered a distinct pharmacological interaction consistent with functional receptor crosstalk. In situ proximity ligation assays confirmed the formation of CB₁R/GPR55 receptor complexes in co-transfected cells. The protective effects were evident even under serum-containing conditions, suggesting that serum components may provide basal receptor activation. In serum-free conditions, selective activation of CB₁R with arachidonyl-2'-chloroethylamide (ACEA) or GPR55 with CID1792197 enhanced cell survival, while the CB₁R antagonist SR141716 blocked both ACEA- and CID1792197-induced protection. These cross-antagonistic interactions support the presence of heteromer-dependent signaling. Altogether, our findings identify CB₁R/GPR55 heteromers as novel modulators of OLG resilience and highlight their potential as therapeutic targets for promoting neuroprotection and remyelination in demyelinating diseases such as MS.</p>