The thymus provides a unique microenvironment enabling development and selection of T lymphocytes. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by facilitating negative selection of self-reactive thymocytes and the generation of Foxp3(+) regulatory T cells. Although studies have highlighted the non-canonical nuclear factor κB (NF-κB) pathway as the key regulator of mTEC development, comprehensive understanding of the molecular pathways regulating this process still remains incomplete. Here, we demonstrate that the development of functionally competent mTECs is regulated by the histone deacetylase 3 (Hdac3). Although histone deacetylases are global transcriptional regulators, this effect is highly specific only to Hdac3, as neither Hdac1 nor Hdac2 inactivation caused mTEC ablation. Interestingly, Hdac3 induces an mTEC-specific transcriptional program independently of the previously recognized RANK-NFκB signaling pathway. Thus, our findings uncover yet another layer of complexity of TEC lineage divergence and highlight Hdac3 as a major and specific molecular switch crucial for mTEC differentiation.