In Drosophila and Caenorhabditis elegans, kinase suppressor of ras (KSR) positively modulates Ras/Raf-mitogen-activated protein kinase (MAPK) signaling. The precise signaling mechanism of mammalian KSR1 and its role in Ras-mediated transformation, however, remain uncertain. To gain insight into KSR1 function in vivo, we generated mice homozygous null for KSR1. ksr1−/− mice are viable and without major developmental defects. However, an unusual disorganized hair follicle phenotype manifest in epidermal growth factor receptor knockout mice is recapitulated in ksr1−/− mice, providing genetic support for the notion that epidermal growth factor receptor, Ras, and KSR1 are on the same signaling pathway in mammals. Furthermore, ksr1−/− mice allow for the definition of KSR1-dependent and -independent mechanisms of c-Raf-1 activation. In embryonic fibroblasts, epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate activated the MAPK cascade to a similar extent, yet only c-Raf-1 activation by epidermal growth factor depended on KSR1. Moreover, whereas the genesis of polyomavirus middle T antigen (MT)-driven mammary cancer appears independent of KSR1, KSR1 is obligate for v-Ha-ras-mediated skin tumor formation. The growth of MT-driven mammary tumor was moderately slowed in ksr1−/− mice, however, consistent with a decreased rate of proliferation of ksr1−/− cells (T cells and embryonic fibroblasts). Nonetheless, all ksr1−/− animals succumbed to mammary cancer. In contrast, papilloma formation in Tg.AC mice, resulting from skin-specific v-Ha-ras expression, was completely abrogated in the ksr1−/− background. Hence, MT-driven mammary tumor genesis, which is signaled through src and phosphatidylinositol 3′-kinase, appears KSR1 independent, whereas v-Ha-ras-mediated skin cancer, signaled through the Raf-1/MAPK cascade, requires KSR1. These results suggest KSR1 may represent a therapeutic target for Ras/MAPK signaling of human tumorigenesis.