We showed previously that the addition to cultured oligodendrocytes (OLs) of multivalent carbohydrate in the form of liposomes containing the two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and cerebroside sulfate (Sulf), or galactose conjugated to bovine serum albumin caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface. Multivalent carbohydrate also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the carbohydrate with the OL surface transmits a transmembrane signal to the cytoskeleton. In the present study we show that inhibition of GSL synthesis with fumonisin B1 prevents clustering of MBP in GalC/Sulf-negative oligodendrocytes, suggesting that GSLs are required for the effect. Because the effects of multivalent carbohydrate resemble those caused by the addition of anti-GalC/Sulf antibodies to OLs and because GalC and Sulf can interact with each other by trans carbohydrate–carbohydrate interactions across apposed membranes, these results support the conclusion that the OL receptor for GalC/Sulf in liposomes is GalC/Sulf in the OL membrane. Inhibition of MBP expression using MBP siRNA inhibited GalC clustering, suggesting that MBP is required for the effect. We also investigate the signal transduction pathways involved using a number of enzyme inhibitors. These indicated that the Akt and p42/p44 MAPK pathways, Rho GTPases, and GSK-3β are involved, consistent with their known involvement in regulation of the cytoskeleton. These interactions between GalC/Sulf-containing liposomes and the OL membrane may mimic interactions between GalC/Sulf-enriched signaling domains when OL cell membranes or the extracellular surfaces of compact myelin come into contact. © 2008 Wiley-Liss, Inc.