The activities of the Raf kinase family proteins control extracellular signal-regulated kinase (ERK) activation in many aspects of cellular responses. However, the relative contributions of individual isozymes to cellular functions including T cell responses are still unclear. In addition to Raf-1, another Raf family kinase, B-Raf, is expressed in murine thymocytes and peripheral T cells, and its activation was induced by TCR stimulation. Here, we investigated the function of B-Raf in development of T cells by generating chimeric mice in which a T cell-compromised host was reconstituted with fetal liver-derived cells from embryonic lethal B-Raf-deficient mice. Although B-Raf was dispensable for normal T cell lineage differentiation at the CD4(-)CD8(-) double-negative stage, thymocytes in the chimeric mice derived from B-Raf(-/-) cells exhibited a drastic arrest of differentiation at the CD4(+)CD8(+) double-positive stage, suggesting that B-Raf is crucial for T cell development, especially for the transition to CD4(+) and CD8(+) single-positive thymocytes. Regarding intracellular signaling, we found that activation of ERK following TCR stimulation was impaired in the thymocytes from the chimeric mice. In conclusion, we present first evidence for the important role of B-Raf-mediated signaling in T cell development.