Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL-60. To further define the potential for HL-60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low-dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL-60 activation. In combination with activation, low-dose irradiation abrogated replication while sparing the candidacidal effects of the HL-60 cells. Infusion of irradiated, activated HL-60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL-60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.