The autoimmune regulator, AIRE, induces the transcription of thousands of peripheral tissue self-antigens (PTAs) in thymic epithelial cells (TECs). This activity mediates self-tolerance via the clonal deletion of auto-reactive thymocytes expressing high-affinity TCRs recognising such self-antigens and/or the selection of FoxP3⁺ regulatory T cells. The molecular mechanisms by which AIRE orchestrates such broad, tolerogenic transcription of PTAs in TECs remain poorly defined. Post-translational modifications of histone proteins, such as acetylation, play an important role in the transcriptional regulation of genes. HBO1 is a histone acetylase that can promote/repress transcription at a range of loci, prompting us to examine whether HBO1 plays a role in the epigenetic regulation of transcription of PTAs in TECs. We took a conditional genetic deletion approach, ablating a floxed allele of Hbo1 specifically in TECs using Foxn1^Cre. Young Foxn1^Cre;Hbo1^fl/fl mice exhibited thymic hypo-cellularity with a significant reduction in overall TEC numbers. The medullary TEC subset showed a substantial reduction, coincident with an increase in the cortical TEC subset. AIRE-positive medullary TECs were diminished in Foxn1^Cre;Hbo1^fl/fl mice and the remaining cells showed a reduced level of AIRE expression. Additionally, we observed increased T cell activation in young Foxn1^Cre;Hbo1^fl/fl mice and lymphocytic infiltration of the lung in older mice, suggesting that tolerance had been perturbed. RNAseq analysis revealed that the deletion of Hbo1 lead to a significant decrease in the expression of PTAs in the mTEC^high subset. Comparisons to Aire^-/- mTECs showed a high similarity between AIRE and HBO1 target genes implying a role for histone 3 lysine 14 acetylation mediated by HBO1 for establishing a normal thymic microenvironment, normal AIRE expression/function and the establishment of self-tolerance.