Mutations of the human B-RAF gene are detected in ∼8% of cancer samples, primarily in cutaneous melanomas (70%). The most common mutation (90%) is a valine-to-glutamic acid mutation at residue 600 (V600E; formerly V599E according to previous nomenclature). Using a Cre/Lox approach, we have generated a conditional knock-in allele of ^V600EB-raf in mice. We show that widespread expression of ^V600EB-Raf cannot be tolerated in embryonic development, with embryos dying ∼7.5 dpc. Directed expression of mutant ^V600EB-Raf to somatic tissues using the IFN-inducible Mx1-Cre mouse strain induces a proliferative disorder and bone marrow failure with evidence of nonlymphoid neoplasia of the histiocytic type leading to death within 4 weeks of age. However, expression of mutant B-Raf does not alter the proliferation profile of all somatic tissues. In primary mouse embryonic fibroblasts, expression of endogenous ^V600EB-Raf induces morphologic transformation, increased cell proliferation, and loss of contact inhibition. Thus, ^V600EB-Raf is able to induce several hallmarks of transformation in some primary mouse cells without evidence for the involvement of a cooperating oncogene or tumor suppressor gene. (Cancer Res 2005; 65(24): 11493-500)