Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as α-galactosylceramide (αGalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-γ or IL-4 production by these cells. We hypothesized that αGalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in αGalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, αGalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells.