During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells. The molecular signals that promote this process, however, are not well defined. One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn. In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed. Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library. Coimmunoprecipitation and in vitro binding assays verified that p190 RhoGAP bound to the Fyn SH2 domain. Phosphorylation of p190 required active Fyn tyrosine kinase and was increased threefold upon differentiation of primary oligodendrocytes. Moreover, complex formation between p190 and p120 RasGAP occurred in differentiated oligodendrocytes. p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio. Indeed, expression of dominant negative Rho in primary oligodendrocytes caused a hyperextension of processes. Conversely, constitutively activated Rho caused reduced process formation. These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 62–78, 2001