Medullary thymic epithelial cells (mTECs) ectopically express a diversity of peripheral tissue-restricted antigens (PTAs) and provide unique cues for the expansion, maturation and selection of a repertoire of functionally diverse T lymphocytes. Genetic deletion of all mature microRNAs in thymic epithelial cells (TECs) results in premature thymic involution, progressive disorganisation of the thymic epithelium, and alteration in thymic T cell lineage commitment, consequently eliciting autoimmune disorders. In the present study, we identified that microRNA-449a (miR-449a), a member of miR-449 cluster, regulated mTEC differentiation. Expression of miR-449a was induced by RANK ligand in mouse fetal thymus. In in vitro studies, overexpression of miR-449a induced thymic epithelial progenitor cells (TEPCs) differentiation into mature mTECs. Despite abundant expression of miR-449a in developing thymus, miR-449a-mutant mice exhibited normal thymic development. This might be partially due to in miR-449a-mutant thymus the up-regulation of miR-34a which shared similar seed sequence with miR-449a. However, thymic expression of miR-449/34 sponge which was able to neutralize the function of miR-449/34 family members significantly reduced the number of mature Ly51-MHCIIhi mTECs. Taken together, our data suggested that miR-449a modulated mTEC differentiation, and members of miR-34 cluster functioned redundantly to rescue miR-449a deficiency in thymus development.