Bone marrow (BM) derived dendritic cells (DCs) cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) have been used to generate antitumor immune responses. The cytokine Fit3 ligand (Flt3L) also has been shown to generate BM DCs. We sought to determine if DCs generated by using Flt3L then matured with lipopolysaccharide (LPS) could lead to DCs with in vivo anti-acute myelogenous leukemia (anti-AML) activity. LPS and tumor necrosis factor α (TNF-α) are effective agents for maturing DCs; however, they have potential in vivo toxicities. Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpGs) are considered relatively nontoxic, potent activators of DC function and maturation in vitro and in vivo. We investigated whether CpGs would be comparable to TNF-α or LPS for the maturation of GM-CSF/ IL-4-generated DCs. DCs cultured with GM-CSF/IL-4 and matured with TNF-α, LPS, or CpG produced a greater allogeneic T-cell response compared with Flt3L/LPS-generated DCs. All 4 distinct DC types were pulsed with AML-lysate and administered before tumor challenge produced an increase in the total number of splenic anti-AML-specific cytotoxic T-lymphocyte precursors and led to significantly (P ≤ .0001) improved survival compared with nonvaccinated controls. GM-CSF/IL-4/LPS was superior to Flt3L/LPS for generating anti-AML effects in vivo. Whereas TNF-α was comparable to LPS in conferring on GM-CSF/IL-4 DCs anti-AML effects in vivo, CpGs were superior to LPS. These data have important clinical implications and are the first to show that Flt3L-generated DCs can provide antitumor protection and that nontoxic agents such as CpGs and Flt3L may be useful in the clinical development of DC vaccines.