The thymus is an organ in which T cells develop and their antigen recognition repertoire is established. 1 In the three-dimensional microenvironment composed of thymic stromal cells, immature T cells (called thymocytes) undergo stepwise developmental processes, including differentiation, proliferation, and cell fate determination in order to give rise to mature T cells expressing a diverse T cell receptor (TCR) repertoire. 2 The thymus parenchyma is subdivided into two regions, the cortex and medulla, wherein distinct subsets of thymic epithelial cells (TECs) form a reticular meshwork that houses developing thymocytes. 3,4 The cortex is the outer region with cortical TECs (cTECs) and thymocytes of immature stages, while the medulla is the inner region and is characterized by medullary TECs (mTECs) and mature thymocytes (Figure 1A). TECs play an essential role in T cell development, providing various signals in support of the survival, proliferation, migration, differentiation, and repertoire selection of thymocytes.Early T-cell progenitors (ETPs) from the fetal liver or adult bone marrow differentiate into CD4 − CD8 − (double negative, DN) thymocytes in the thymic cortex. Guided by cTECs, DN thymocytes are committed to the T-cell lineage and undergo rearrangements of the genes encoding the TCR. [3][4][5] In the adult thymus, ETPs arrive at the cortico-medullary junction (CMJ) where blood vessels are enriched, and developing DN thymocytes migrate through the cortex toward

Medullary thymic epithelial cells (mTECs) help shape the thymic microenvironment for T cell development by expressing a variety of peripheral tissue-restricted antigens (TRAs). The self-tolerance of T cells is established by negative selection of autoreactive T cells that bind to TRAs. To increase the diversity of TRAs, a fraction of mTECs terminally differentiate into distinct subsets resembling atypical types of epithelial cells in specific peripheral tissues. As such, thymic tuft cells that express peripheral tuft cell genes have recently emerged. Here we show that the transcription factor Sox4 is highly expressed in mTECs and is essential for the development of thymic tuft cells. Mice lacking Sox4 specifically in TECs had a significantly reduced number of thymic tuft cells with no effect on the differentiation of other mTEC subsets, including Aire + and Ccl21a + mTECs. Furthermore, Sox4 expression was diminished in mice deficient in TEC-specific lymphotoxin β receptor (LTβR), indicating a role for the LTβR-Sox4 axis in the differentiation of thymic tuft cells. Given that Sox4 promotes differentiation of peripheral tuft cells, our findings suggest that mTECs employ the same transcriptional program as peripheral epithelial cells. This mechanism may explain how mTECs diversify peripheral antigen expression to project an immunological self within the thymic medulla.