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The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T-cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of beta 5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8(+) T cells. A point mutation in this genome element results in a defect in beta 5t expression and CD8(+) T-cell production in mice. The results reveal a Foxn1-beta 5t transcriptional axis that governs CD8(+) T-cell production in the thymus.