T hree distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-α upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-α/β–producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin^–, HLA-DR^int, IL-3Rα^hi, CD45RA^hi, CD11c^–, CD13^–, and CD33^lo) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR^int, CD11c⁺, CD13⁺, CD33⁺), and the other represented HLA-DR^hi CD11c⁺ mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-Tα, λ-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.