The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cell differentiation, but the signaling mechanisms by which it is activated during this process are largely unknown. Cdo is an immunoglobulin superfamily member that functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that the Cdo intracellular region interacts with JLP, a scaffold protein for the p38alpha/beta MAPK pathway. Cdo, JLP, and p38alpha/beta form complexes in differentiating myoblasts, and Cdo and JLP cooperate to enhance levels of active p38alpha/beta in transfectants. Primary myoblasts from Cdo(-/-) mice, which display a defective differentiation program, are deficient in p38alpha/beta activity, and the expression of an activated form of MKK6 (an immediate upstream activator of p38) rescues the ability of Cdo(-/-) cells to differentiate. These results document a novel mechanism of signaling during cell differentiation: the interaction of a MAPK scaffold protein with a cell surface receptor.