Hexokinases (HK) are enzymes that catalyze the first step of glycolysis – the phosphorylation of glucose to glucose-6-phosphate. Hexokinase 3 (HK-3), one of four mammalian HK isoforms, is detected in hematopoietic cells and tissues, especially in cells of myeloid lineage. Previous studies have shown that after myeloid cells undergo CRISPR/Cas9-mediated HK-3 gene disruption and differentiation to neutrophil-like cells, loss of HK-3 leads to no effect on glycolysis, but an increased cell death phenotype during differentiation to neutrophil-like cells. This study aimed to examine if HK-3 gene knockout causes a similar cell death phenotype in monocyte/macrophages cells, which are of myeloid origin. To test directly, THP-1 cells, which are monocyte-like cells that can be differentiated to macrophage-like cells, underwent CRISPR/Cas9-mediated HK-3 gene knockout via electroporation. This is a method that uses electric pulses to make pores within the cell and nuclear membrane. PCRs and DNA sequencing comparing wild-type THP-1 cells and HK-3-null THP-1 cells were done to confirm successful knockout. The HK-3-null THP-1 cells then underwent 48-hour, 250 nM PMA-induced differentiation to macrophage-like cells and were then stained with Trypan Blue to compare viability of wild-type macrophage-like cells and HK-3-null macrophage-like cells. No apparent difference was found in cell viability between wild-type macrophage-like cells and HK-3-null macrophage-like cells during differentiation and over six days post-differentiation. Cell function was not assessed. This suggests that further work should be to: (1) confirm these results by repeating the HK-3-null in THP-1 cell line process and remeasuring cell viability after differentiation, (2) perform the HK-3-null process in other cell lines to differentiate into other cells of myeloid origin to determine if cell death phenotype appears, and (3) determine if loss of HK-3 alters viability under culture conditions mimicking low oxygen conditions, such as found in hypoxic tissue areas during bacterial infection. Understanding cell viability differences from loss of HK-3 could give insight into the potential regulatory function of HK-3 during myeloid hematopoiesis.