Differentiation of oligodendrocytes results in the formation of the myelin sheath, a dramatic morphological alteration that accompanies cell specialization. Here, we demonstrate that changes in the extracellular micro environment may regulate these morphological changes by altering intracellular vesicular trafficking of myelin sheet-directed proteins. The data reveal that fibronectin, in contrast to laminin-2, decreased membrane-directed transport of endogenous NCAM 140 and the model viral protein VSV G, both proteins normally residing in the myelin membrane. The underlying mechanism relies on an integrin-mediated activation of PKC, which causes stable phosphorylation of MARCKS. As a result, dynamic reorganization of the cortical actin cytoskeleton necessary for the targeting of vesicular trafficking to the myelin sheet is precluded, a prerequisite for morphological differentiation. These data are discussed in the context of the demyelinating disease multiple sclerosis, i.e., that leakage of fibronectin across the blood-brain barrier may impede myelination by interference with intracellular myelin sheet-directed membrane transport. (c) 2006 Elsevier Inc. All rights reserved.