Dendritic cells (DCs) are recognized as key regulators of the immune system. Active DC immunization protocols are quickly obtaining interest as an alternative therapeutic approach in acute myeloid leukemia patients. Despite apparent progress in DC-based immunotherapy, some discrepancies were reported in generating potent DCs and their source. In addition to monocytes, DCs can be differentiated from leukemic blasts of acute myeloid leukemia (AML) patients (AML-DC) possessing the ability of stimulating anti-leukemic immune response. In this study, we differentiated peripheral blood blasts of 16 out of 20 AML patients in vitro in the presence of GM-CSF and IL-4 into immature AML-DC. Then, DCs matured using different combinations of Toll-like receptor (TLR) ligands to obtain functional DCs as demonstrated by cell morphology, immunophenotype, and functional activity. Autologous cytotoxic T cell induction of matured DCs was evaluated in four patients and compared with immature counterparts. Our results showed that although the TLR3 agonist (Poly I:C) has a synergistic effect on the TLR4 agonist (lipopolysaccharide, LPS), the addition of the TLR7/8 agonist (R848) is necessary to reinforce the effect of LPS or LPS + POLY(I:C) to produce efficient DCs with the higher level of IL-12 (30 to 90 times). Such DCs activate allogeneic T cells and effectively prime autologous cytotoxic T cells in vitro. In contrast, FSL-1 as a TLR2/6 agonist has a negative effect on LPS + Poly(I:C) and LPS + R848 to produce IL-12. Thus, DCs prepared using a maturation mixture including a TLR7/8 agonist may be used as a potential tool for DC-based immunotherapy purposes in leukemic patients.