A key process in the development of T lymphocyte in the thymus is T‐cell receptor (TCR) selection. It is controlled by complex signaling pathways that contain redox‐sensitive molecules. However, the redox status early after TCR selection and how redox regulators promote the survival of post‐selected DP thymocytes has not been directly addressed. The present study demonstrated that the transition from pre‐ to post‐selected double‐positive (DP) stages was accompanied with an increase of reactive oxygen species (ROS) and a transient surge in the expression of a variety of redox regulators. Among them, the thioredoxin (Trx)1/thioredoxin reductase (TrxR)1 system was found to be critically involved in the regulation of cell survival of DP thymocytes, especially that of post‐selected CD69⁺ subset, as its inhibition caused a specific reduction of these cells both in vitro and in vivo, most likely owing to increased apoptosis. Suppression of the glutathione‐dependent redox system, on the other hand, showed no obvious impact. Biochemically, treatment of DP thymcoytes with TrxR1 inhibitor alone or in conjunction with anti‐CD3 resulted in enhanced phosphorylation of redox‐sensitive ASK‐1, JNK and p38 MAPK, and upregulated expression of Bim. Taken together, the data presented here suggest that the timely upregulation of Trx1/TrxR1 and the active control of intracellular redox status is critical for the survival of thymocytes during and short after positive selection.