The thymus is the organ in which T lymphocytes mature. Thymocytes undergo exhaustive selection processes that require interactions between the TCRs and peptide-HLA complexes on thymus antigen-presenting cells. The thymic peptide repertoire associated with HLA molecules must mirror the peptidome that mature T cells will encounter at the periphery, including peptides that arise from tissue-restricted antigens. The transcriptome of specific thymus cell populations has been widely studied, but there are no data on the HLA-I peptidome of the human thymus. Here, we describe the HLA-I-bound peptide repertoire from thymus samples, showing that it is mostly composed of high-affinity ligands from cytosolic and nuclear proteins. Several proteins generated more than one peptide, and some redundant peptides were found in different samples, suggesting the existence of antigen immunodominance during the processes that lead to central tolerance. Three HLA-I ligands were found to be derived from proteins expressed by stromal cells, including one from the protein TBATA (or SPATIAL), which is present in the thymus, brain and testis. The expression of TBATA in medullary thymic epithelial cells has been reported to be AIRE dependent. Thus, this report describes the first identification of a thymus HLA-I natural ligand derived from an AIRE-dependent protein with restricted tissue expression. CONCLUSIONS We present the first description of the HLA-I-bound peptide repertoire from ex vivo thymus samples. This repertoire is composed of standard ligands from cytosolic and nuclear proteins. Some peptides seem to be dominantly presented to thymocytes in the thymus. Most importantly, some HLA-I associated ligands derived from proteins expressed by stromal cells, including one peptide, restricted by HLA-A*31:01, arising from an AIRE-dependent protein with restricted tissue expression.