CD4+ CD45RO+ T cells could mature freshly isolated human plasmacytoid dendritic cells (PDC) in a superantigen-driven culture in a similar way to recombinant interleukin-3 (IL-3). Mature PDC expressed significantly higher levels of inducible costimulator ligand (ICOS-L), but similar levels of CD80 and CD86, when compared to mature monocyte-derived DC (moDC). We therefore directly compared the capacities of mature PDC and moDC to activate T cells. A similar T helper type 1 (Th1)/Th2 pattern of cytokines was generated in both systems, but significantly higher levels of IL-3, IL-4 and IL-10 were induced by PDC. In T cells interacting with PDC, the ICOS/ICOS-L costimulatory pathway played a pre-eminent role in the generation of IL-3 and IL-10, CD28 was central to the induction of IL-2, and both pathways were equally important for the generation of other cytokines. In cocultures with moDC, the CD28 pathway was dominant over ICOS under all circumstances, except for the ICOS-mediated release of IL-10. In general, our data demonstrate an eminent role of ICOS in the interaction of T cells with PDC, and thus modify the current paradigm of CD28 dominance for the costimulation of T cells interacting with professional antigen-presenting cells. In particular, our data highlight the role of ICOS in the generation of IL-3, a factor central to the biology of human PDC.