The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (alpha-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescenceactivated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of alpha-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the alpha-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the alpha-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and 8, respectively. In alpha-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.