The rigorous process of thymocyte selection is aimed at producing a T cell repertoire capable of responding to a broad range of foreign antigens, but tolerant to self. Only a small percentage of thymocytes entering the selection process emerge from the thymus as mature T cells. Survival and death signals through the T cell receptor (TCR) largely dictate thymocyte fate. Thymocytes with auto-reactive TCRs undergo negative selection and are signaled to die by apoptosis. The Nr4a family of nuclear receptors and the Bcl-2 protein family are critical mediators of thymocyte cell death. Here, we investigate the mechanism by which Nr4a receptors translocate from the nucleus to the mitochondria and induce apoptosis through the mitochondrial death pathway. Through mutagenesis of Nr4a family member Nor-1, we identify regions in the N-terminus of the protein critical for mitochondrial localization. Nr4a proteins have been proposed to induce apoptosis by binding Bcl-2 and converting it to a pro-apoptotic effector through exposure of its BH3 domain. We find that thymocytes expressing a Bcl-2 BH3 mutant transgene exhibit increased resistance to TCR-mediated apoptosis in vivo, providing support for Bcl-2 having pro-apoptotic function during negative selection. While death by negative selection is important for inhibiting the production of autoreactive T cells, it remains unclear whether it is necessary for preventing the development of autoimmune disease. Here, we find that Bcl-2 BH3 mutant transgenic mice accumulate activated T cells over time, culminating in the development of multi-organ autoimmunity and early lethality. These data provide new evidence for a crucial role for negative selection in preventing autoimmunity. Aberrant survival signals during thymic selection can lead to the development of T cell malignancies. PTEN, a negative regulator of the PI3-kinase pathway, plays an important role in safeguarding against this fate and in its absence, DP thymocytes form premalignancies that progress to T cell lymphomas. Through the analysis of these premalignancies, we identify two miRNAs that act to suppress malignant transformation by inhibiting proliferation signals downstream of the TCR. Overall, this work provides new insight into the life and death signals important for efficient thymocyte selection and demonstrates how study of their dysregulation in disease can identify novel therapeutic targets.