T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in mCat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFP-LC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.