We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing FIt3L (C3Lt-Flt3L) and GM-CSF (C3L5GMCSF) by injecting 1 x 104 cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 x 103 cells; or low, 1 x 103 cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.