Acute myeloid leukemia (AML) is an aggressive malignancy with limited treatment options. Enhancing natural killer (NK) cell functionality through artificial antigen-presenting cells (aAPCs) represents a promising immunotherapeutic strategy. This study evaluates the potential of genetically modified K562 cells, expressing CD137L and CD86, to enhance NK cell-mediated cytotoxicity against AML cell lines (HL-60, KG-1, and THP-1). Lentiviral transduction was used to generate aAPCs, confirmed by PCR, RT-PCR, and flow cytometry. Cord NK cells and the NK-92 cell line were co-cultured with aAPCs, and their cytotoxicity against cell lines was assessed using 7-AAD staining. The ability of transduced K562 cells to substitute for interleukin-15 (IL-15) was also evaluated. These cells significantly enhanced NK cell-mediated cytotoxicity, with greater effects observed at higher effector-to-target (E:T) ratios. The aAPCs partially replaced IL-15 in activating cord blood NK cells but were ineffective for NK-92 cells. The aAPCs effectively enhance NK cell cytotoxicity and may reduce cytokine dependence in therapeutic applications. These findings highlight the potential of aAPCs to improve NK cell-based immunotherapies.