Clonal selection of T lymphocytes is essential for establishing self/non-self discrimination of immune recognition. It is known that cell surface signals such as avidity and valency of TCR-ligand interactions influence the fate of individual thymocytes, founding a primary repertoire of T cells. However, intracellular signals that govern positive and negative selection in the thymus have been unclear. The present study using the retroviral gene transfer technique shows that MKK1 activation in developing T cells is sufficient for providing positive selection signals. We also show that the MKK6-p38 signaling pathway is critically involved in inducing negative selection of thymocytes. These results suggest that intracellular signals through different MAP kinase cascades selectively guide positive and negative selection of T lymphocytes.