AbstractT cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen‐activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v‐Raf under the control of the p56lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)‐mediated signaling events as assessed by the inhibition of mitogen‐induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8+ thymocytes in female mice doubly transgenic for DN‐Raf and a class I‐restricted H‐Y TCR. In contrast, the differentiation of double‐positive thymocytes to single‐positive thymocytes was enhanced in H‐YTCR transgenic mice expressing constitutively active Raf (v‐Raf). Thus, Raf regulates positive selection in the thymus.