The key interaction in the adaptive immune system's response to pathogenic challenge occurs at the interface between APCs and T cells. Families of costimulatory and coinhibitory molecules function in association with the cytokine microenvironment to orchestrate appropriate T cell activation programs. Recent data have demonstrated that the Notch receptor and its ligands also function at the APC:T interface. In this study, we describe synthetic small interfering RNA (siRNA) sequences targeting the human Notch ligands Delta1, Jagged1 and Jagged2. Transfection of these siRNAs into human primary CD4(+) T cells and monocyte-derived dendritic cells leads to knockdown of endogenous Notch ligand message. Knockdown of any one of these three Notch ligands in dendritic cells enhanced IFN-gamma production from allogeneic CD4(+) T cells in MLR. In contrast, Delta1 knockdown in CD4(+) T cells selectively enhanced production of IFN-gamma, IL-2, and IL-5 in response to polyclonal stimulation, while Jagged1 or Jagged2 knockdown had no effect. Strikingly, blockade of Notch cleavage with a gamma secretase inhibitor failed to affect cytokine production in this system, implying that Delta1 can influence cytokine production via a Notch cleavage-independent mechanism. These data show for the first time that the Notch pathway can be targeted by siRNA, and that its antagonism may be a unique therapeutic opportunity for immune enhancement.