Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα-chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll-like receptor (TLR) ligand activation of TCR-transgenic murine dNKT cells. IFN-γ production by dNKT cells required dendritic cells (DC), cell-to-cell contact and presence of TLR ligands. TLR-stimulated DC activated dNKT cells to secrete IFN-γ in a CD1d-, CD80/86- and type I IFN-independent manner. In contrast, a requirement for IL-12p40, and a TLR ligand-selective dependence on IL-18 or IL-15 was observed. TLR ligand/DC stimulation provoked early secretion of pro-inflammatory cytokines by both CD62L and CD62L dNKT cells. However, proliferation was limited. In contrast, TCR/co-receptor-mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co-receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory. This article is protected by copyright. All rights reserved.