E2A and HEB are basic helix-loop-helix transcription factors essential for T cell development. Complete inhibition of their activities through transgenic overexpression of their inhibitors Id1 and Tal1 leads to a dramatic loss of thymocytes. Here, we suggest that bHLH proteins play important roles in establishing thresholds for pre-TCR and TCR signaling. Inhibition of their function allows double-negative cells to differentiate without a functional pre-TCR, while anti-CD3 stimulation downregulates bHLH activities. We also find that the transcription factor NF-κB becomes activated in transgenic thymocytes. Further activation of NF-κB exacerbates the loss of thymocytes, whereas inhibition of NF-κB leads to the rescue of double-positive thymocytes. Therefore, we propose that E2A and HEB negatively regulate pre-TCR and TCR signaling and their removal causes hyperactivation and apoptosis of thymocytes.