Primary cilia are ubiquitous hair-like organelles, usually projecting from the apical cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. After detecting primary cilia on the thymic epithelial cell (TEC) expressed transforming growth factor β (TGF-β) receptor in the basal body, we established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88 TEC null mutant) to clarify their precise role in thymic organogenesis and T cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the “thymic synapse” of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-β signaling cascade, increasing the number of single positive thymocytes.