CD3(+)CD56(-), CD4 and CD8 double negative T (DNT) cells comprise 1-3% of peripheral blood (PB) mononuclear cells. Their role in tumor immunity remains largely unknown due to their limited numbers and lack of effective methods to expand them. Here we developed a novel protocol by which DNT cells can be expanded ex vivo to therapeutic levels in 2 weeks from 13 of 16 acute myeloid leukemia (AML) patients during chemotherapy-induced complete remission. The expanded DNT cells expressed similar or higher levels of interferon-γ and tumor necrosis factor-α and Granzyme B as that seen in bulk activated CD8T cells from the same patient but significantly higher levels of perforin. The expanded DNT cells could effectively kill both allogeneic and autologous primary CD34(+) leukemic blasts isolated from PB of AML patients in a perforin-dependant manner. These results demonstrate, for the first time, that DNT cells from AML patients can be expanded ex vivo even after intensive chemotherapy, and are effective at killing both allogeneic and autologous primary leukemic blasts. These findings warrant studies further exploring the potential of DNT cells as a novel adjuvant immunotherapy to decrease the risk of relapse in patients with AML and, perhaps, other cancers.