Invariant Natural Killer T (iNKT) cells are CD1d restricted innate lymphoid cells with an invariant T cell receptor (TCR) alpha chain gene rearrangement (Valpha24-Jalpha18 in human and Valpha14-Jalpha18 in mouse). iNKT cells play a pivotal role in anti-tumor immune responses via cytokine mediated transactivation of various cells which mediate innate and adaptive immune responses. Here we describe, to our knowledge for the first time, the generation of long-term mouse spleen derived iNKT cell lines. We found that dendritic cells (DC) derived from the D1 line, but not Mf4/4 macrophages, loaded with the artificial iNKT cell ligand alpha-Galactosylceramide (alphaGalCer) could be employed to expand iNKT cells in vitro. Furthermore, exogenously added IL-7, but not IL-2 or IL-15 had a pronounced additive effect on iNKT cell expansion. Using this method up to 10(8) iNKT cells could be obtained from one spleen within 12 to 14 weeks, and cell lines could be continued for up to 24 months. Importantly, the iNKT cell lines had retained the capacity to swiftly secrete substantial amounts of both T helper (Th) 1 and Th2 cytokines upon activation. In conclusion we have generated iNKT cell lines with high yields that can be maintained for up to 24 months, by repeated stimulation using alpha-GalCer loaded D1.DC and IL-7. These in vitro expanded iNKT cells preserved the capacity to swiftly produce both Th1 and Th2 type cytokines and are currently being utilized in pre-clinical adoptive transfer models to identify and optimize the characteristics of therapeutically effective iNKT cells in an anti-tumor setting.