NKT cell can modulate the immune response through the production of type 1 T helper (Th1), Th2, or even Th17 cytokines and serve as a good target for immunotherapy. Selective enhancement of either Th1- or Th2-cytokine production upon stimulation may better control immune-mediated diseases according to the respective immunopathology. By employing a co-culture of NKT cells with differently treated dendritic cells (DC) and quantifying cytokines in the culture supernatants, we have developed novel methods to enhance either IFN-γ or IL-4 production by NKT cells. When α-galactosylceramide-loaded DCs were pre-treated with IL-4 or IFN-γ and then co-cultured with NKT cells, the enhanced production of IFN-γ or IL-4 by NKT cells was respectively induced, implying that NKT cells could produce a cytokine of the opposite response to the cytokine used for pre-treatment of the DCs. Dynamics of inhibitory ligand expression on DCs appear to be involved in this phenomenon. Utilization of negative feedback regulation may expand the utility of NKT cells for therapy for tumors, infectious diseases, and autoimmunity.