To find a feasible method for the stimulation of tumor-draining lymph node (TDLN) cells in preparation for use in the clinic, the CTL activity of TDLN cells induced by different stimuli [IL-2 alone, IL-2 + autologous tumor antigen (atAg), IL-2 + GM-CSF + IL-4 + atAg] was measured by maximal LDH enzyme release. The mechanisms were explored by the observation of morphology and the detection of CD83+ TDLN cells. The expansion of TDLN cells by IL-2 + GM-CSF + IL-4 + atAg was significantly higher than that by IL-2 alone or IL-2 + atAg (p<0.01). Antitumor CTL activity of TDLN cells induced by IL-2 + GM-CSF + IL-4 + atAg was significantly higher than those of other groups. The number of CD83+ cells within the TDLN population treated with IL-2 + GM-CSF + IL-4 + atAg was significantly elevated. The method of stimulating TDLN cells by IL-2 + GM-CSF + IL-4 + atAg was better than the stimulation with IL-2 or IL-2 + atAg. TDLN cells induced by IL-2 + GM-CSF + IL-4 + atAg produced more dendritic cells (DCs). In our study, we established a system that T cells and DCs were stimulated together ex vivo, which was easy to conduct and produce promising results. It provided a new method for improving TDLN cell antitumor activity which might be used in the clinical cancer therapy.