The generation of natural regulatory T cells (nTregs) is crucial for the establishment of immunologic self-tolerance and the prevention of autoimmunity. Still, the origin of nTregs and the mechanisms governing their differentiation within the thymus are poorly understood, particularly in humans. It was recently shown that conventional dendritic cells (cDCs) in human thymus were capable of inducing nTreg differentiation. However, the function of plasmacytoid DCs (pDCs), the other major subset of thymic DCs, remains unknown. Here we report that pDCs resident in the human thymus, when activated with CD40 ligand (CD40L) plus interleukin-3, efficiently promoted the generation of CD4⁺CD25⁺Foxp3⁺ nTregs from autologous thymocytes. The progenitors of these nTregs were selectively found within CD4⁺CD8⁺ thymocytes that had accomplished positive selection, as judged by their CD69^hiTCR^hi phenotype. Supporting the involvement of the CD40-CD40L pathway in pDC-induced nTreg generation, we show that positively selected CD4⁺CD8⁺ progenitors specifically transcribed CD40L in vivo and up-regulated CD40L expression on T-cell receptor engagement, thereby promoting the activation of pDCs. Finally, evidence is provided that nTregs primed by pDCs displayed reciprocal interleukin-10/transforming growth factor-β cytokine expression profiles compared with nTregs primed by cDCs. This functional diversity further supports a nonredundant tolerogenic role for thymic pDCs in the human thymus.