Acute lymphoblastic leukemia (ALL) is believed to be resistant to NK cell-mediated killing. To overcome this resistance, we developed an innovative approach based on NK cell stimulation with Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC). The translation of this approach into the clinic requires the production of high numbers of human pDC. Herein, we show that in vitro differentiation of cord blood CD34+ progenitors in the presence of aryl hydrocarbon receptor antagonists gives rise to clinically relevant numbers of pDC, as about 108 pDC can be produced from a typical cord blood unit. Blocking the aryl hydrocarbon receptor (AHR) pathway significantly increased the yield of pDC. When compared to pDC isolated from peripheral blood, in vitro differentiated pDC (ivD-pDC) exhibited an increased capacity to induce NK cell-mediated killing of ALL. Although ivD-pDC produced lower amounts of IFN-α than peripheral blood pDC upon TLR activation, they produced more IFN-λ2, known to play a critical role in the induction of anti-tumoral NK cell functions. Both TLR-9 and TLR-7 ligands triggered pDC-induced NK cell activation, offering the possibility to use any clinical-grade TLR-7 or TLR-9 ligands in future clinical trials. Finally, adoptive transfer of ivD-pDC cultured in the presence of an AHR antagonist cured humanized mice with minimal ALL disease. Collectively, our results pave the way to clinical-grade production of sufficient numbers of human pDC for innate immunotherapy against ALL and other refractory malignancies.