The neurofibromatosis type 1 (NF1) gene product, neurofibromin, regulates activation of the Ras intracellular signaling pathway in Schwann cells. Schwann cells purified from mouse embryos with null mutations in the Nf1 gene increase expression of the major myelin glycoprotein P0. v-Ras expression in cultured Schwann cells partially mimics loss of Nf1, suggesting a role for Ras in upregulation of P0 expression in Nf1-deficient cells. We tested whether loss of Nf1 alters the ability of Schwann cells to form myelin. No significant changes in myelin formation resulted when Nf1-deficient or v-Ras-expressing Schwann cells were cultured with normal neurons. Yet, in organotypic cultures of neurons, Schwann cells, and fibroblasts without neurofibromin, myelination was dramatically reduced. We suggest that Nf1-dependent signaling cascades in neurons and/or fibroblasts, as well as Schwann cells, are required for normal myelination.