Oncogenic mutations in B-Raf and Kirsten-Ras (K-Ras) are mutually exclusive during human cancer pathogenesis. In an effort to study the biological basis of this epistasis, gene targeting was used to create isogenic sets of human cancer cells differing only in presence or absence of endogenous oncogenic K-Ras or wild-type B-Raf. Whereas cells lacking the K-Ras oncogene were unable to efficiently form xenograft tumors, isogenic cells retaining activated K-Ras but deleted for B-Raf remained highly tumorigenic. Deletion of oncogenic K-Ras failed to reduce the activation state of B-Raf or ERK1/2, despite the requirement of oncogenic K-Ras for tumorigenesis. Genechip analysis revealed numerous genes in which the regulation by oncogenic K-Ras did not require B-Raf. These studies suggest that despite the mutual exclusivity of K-Ras and B-Raf mutations in human cancer and the well-described role for Raf proteins as Ras effectors, B-Raf is dispensable for K-Ras-mediated oncogenesis in a human cancer cell line. Additional studies are required to demonstrate the generalizability of these unexpected findings.