Type I natural killer T (NKT) cells are the most well characterized subset of CD1d-restricted T cells. NKT cells express an invariant αβ TCR and are known for their ability to rapidly produce cytokines following stimulation. Thus, NKT cells can play an important role in antitumor immune responses. Many studies examining patients with cancer have shown that there is a reduction in both NKT cell number and function. These data strongly suggest that an increase in NKT cell number and activity by either adoptive transfer of ex vivo expanded NKT cells or direct in vivo modulation will be an effective therapeutic strategy. A major challenge in developing successful immunotherapy is the difficulty in expanding NKT cells to sufficient amounts. Variation in the quality and quantity of lipid-pulsed DCs typically used for this is a major limitation. To overcome these challenges, we have developed CD1d-based artificial antigen presenting cells (aAPC) that can facilitate the activation and proliferation of human and mouse NKT cells. Development of aAPC is important for its potential clinical value to expand ex vivo NKT cells, as well as its ability to characterize the basic requirements for their activation. In preliminary studies, we have found that CD28 is important for human NKT cell proliferation, whereas stimulation by other co-signaling molecules such as CD44 enhances cytokine production and OX-40L increases cytotoxicity. We hypothesize that the addition of three costimulatory signals will lead to optimal NKT cell responses to cancer, compared to traditional CD28 costimulation. To determine if CD1daAPC can enhance NKT cell mediated anti-tumor responses, we set up cocultures with NKT cells, cancer cell lines in the presence and absence of CD1d-expressing aAPC, and measure cytokine levels by ELISA. While stimulation with cancer cells alone did not induce NKT cell activation, the addition of CD1d-aAPC resulted in cytokine production by NKT cells. Future studies will focus on optimizing the CD1d-expressing aAPC to further enhance NKT cell-mediated anti-tumor immunity.