To evaluate the role of redox homeostasis in differentiation in human promyelocytic leukemia cells (HL-60) induced by isoliquiritigenin (ISL) through modulation of the nuclear erythroid-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway. Morphological changes, cell surface markers CD11b/CD14, and nitroblue tetrazolium (NBT)-reducing ability were used to determine the differentiation of HL-60, and 2,7-dichlorofluorescein was used to detect the level of intracellular reactive oxygen species (ROS). Thiobarbituric acid test was utilised to determine the levels of malondialdehyde production in ISL-treated HL-60. The study determines and presents the redox state of the ratio of reduced/oxidised glutathione as a consequence of progression from differentiation in HL-60. Expression levels of the Nrf2/ARE downstream target genes were determined by quantitative polymerase chain reaction. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitors, apocynin (APO), and diphenyleneiodonium (DPI) were used for the preliminary study to determine the potential downstream targets regulated by NADPH oxidase in ISL-induced HL-60 differentiation. The data showed a strong dose–response relationship between ISL exposure and the characteristics of HL-60 differentiation, namely, morphology changes, NBT reductive activities, and expression levels of surface antigens CD11b/CD14. Intercellular redox homeostasis changes toward oxidation during drug exposure are necessary to support ISL-induced differentiation. The unique expression levels of the Nrf2/ARE downstream target genes in the differentiation of HL-60 recorded a statistically significant and dose-dependent increase (P < 0.05), which were suppressed by NADPH oxidase inhibitor, APO, and DPI. ISL as a differentiation-inducing agent with mechanisms involved in the Nrf2/ARE pathway to modulate intercellular redox homeostasis, and thus, facilitate differentiation.