Abstract We have demonstrated that MHC class II expressing thymocytes can efficiently mediate positive selection of CD4 T cells in mice. Human thymocytes express MHC class II and thymocyte-mediated CD4 T cells selection provides a mechanism for several documented observations made in humans that could not be explained otherwise. We named thymocyte-selected CD4 cells T-CD4 (Thymocyte-selected) and the other E-CD4 (Epithelial cell-selected) to reflect their selection pathway. T-CD4 T cells are distinct and different from E-CD4 T cells by exhibiting the effector function similar to innate T cells. T-CD4 cells can produce both Th1 and Th2 cytokines simultaneously shortly after activation. Remarkably, Stat6 that is critical for Th2 cell differentiation is not required for T-CD4 T cells to produce Th2 cytokines. Interestingly, these characteristics are found in NKT cells that are also selected on thymocytes. Consistent with these observations, development of T-CD4 T cells require SLAM/SAP signal that is essential for NKT but not E-CD4 T cells. To understand the molecular mechanisms and signaling pathways that are involved in T-CD4 T cell generation, we studied the role of ITK and RasGAP. ITK deficient cells were selected more efficiently by thymocytes than thymic epithelial cells. In contrast, RasGAP plays a more important role in the generation of T-CD4 T cells than E-CD4 T cells. Therefore, E- and T-CD4 T cells require different signaling mechanisms for their optimum development. The study was partly supported by NIH.