Effective treatment of acute myeloid leukemia (AML) remains an urgent unmet need. Adoptive transfer of cytotoxic T cells (CTLs) against leukemia-associated antigen (LAA) has strong potential to improve AML treatment. However, the clinical translation of this therapeutic modality is hindered by the difficulty of obtaining large quantities of LAA-specific CTLs. Stimulating naïve T cells using monocyte-derived dendritic cells (MoDCs) loaded with LAA is commonly used for the generation of CTLs. This approach has drawbacks as MoDCs loaded with desired antigen need to be developed repeatedly with multiple steps and have limited growth potential. We have established immortalized human dendritic cells (DC) lines (termed ihv-DCs). Here, we report the successful generation of CTLs by culturing AML patient-derived T cells with our off-the-shelf ihv-DCs that carry HLA-A2-restricted human telomerase reverse transcriptase (hTERT), a known LAA. These CTLs exert a potent cytotoxic activity against leukemia cell lines and primary AML blasts in vitro. Importantly, using a highly clinically relevant PDX model where CTLs (derived from clinical donors) were adoptively transferred into NSG mice bearing patient-derived AML cells (that were partial or full HLA match with the donors), we showed that the CTLs effectively reduced leukemia growth in vivo. Our results are highly translational and provide proof of concept using the novel DC methodology to improve the strategy of adoptive T cell transfer for AML treatment.