A central issue in signal transduction is the physiological contribution of different growth factor-initiated signaling pathways. We have generated knockin mice harboring mutations in the PDGFalpha receptor (PDGFalphaR) that selectively eliminate its capacity to activate PI3 kinase (alpha(PI3K)) or Src family kinases (alpha(Src)). The alpha(PI3K) mutation leads to neonatal lethality due to impaired signaling in many cell types, but the alpha(Src) mutation only affects oligodendrocyte development. A third knockin line containing mutations that eliminate multiple docking sites does not increase the severity of the alpha(PI3K) mutation. However, embryos with mutations in the PI3K binding sites of both PDGFRs (alpha and beta) recapitulate the PDGFalphaR null phenotype. Our results indicate that PI3K has a predominant role in PDGFalphaR signaling in vivo and that RTK-activated signaling pathways execute both specific and overlapping functions during mammalian development.