After positive selection, the newly differentiated single-positive (SP) thymocytes undergo negative selection to eliminate autoreactive T cells, functional maturation to acquire immunocompetence and egress capability. To investigate whether the intracellular reduction/oxidation (redox) balance has an important role on SP maturation, the levels of intracellular reactive oxygen species (ROS) and the expression of proteins that regulate ROS were compared among the four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1-SP4) that represent sequential stages of SP differentiation program. A gradual increase of ROS and a gradual decrease of thioredoxin were revealed along the SP maturation process. The high ROS level at the mature SP stage did not result from a specific enrichment at this stage of natural regulatory T cells and SP thymocytes undergoing negative selection (Helios positive). An increase of ROS in the most mature SP4 cells resulted in enhanced cytokine production upon stimulation, whereas an early increase of ROS in the immature SP1 thymocytes resulted in enhanced apoptosis. Aire(-/-) mice that have defects in negative selection and a developmental blockage at the SP3-SP4 transition showed significantly less ROS in SP3 thymocytes. Thymic epithelial cells that have been shown to promote SP maturation in vitro also increased the ROS level of SP thymocytes. These results suggest that ROS may be involved in promoting the functional maturation of CD4(+) SPs and thymic medullary microenvironment contributes to the pro-oxidant shift of SP thymocytes.