Abstract Background Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator ( AIRE ) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether AIRE influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. Results The comparisons between AIRE wild type ( AIRE WT ) and AIRE KO ( AIRE −/− ) 3D mTEC spheroid formation showed that the absence of AIRE: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells at the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases the strength of mTEC-mTEC adhesion, v) promotes a differential regulation of mTEC classical surface markers, and vi) modulates genes encoding adhesion and other molecules. Conclusions Overall, the results show that AIRE influences the 3D structuring of mTECs when these cells begin the spheroid formation through controlling cell adhesion genes.