Abstract Acute myeloid leukemia (AML) remains a difficult disease to treat disease. In a phase 2 clinical trial in patients with relapsed/refractory AML, combining the hypomethylating agent, azacitidine, with the PD-1 checkpoint inhibitor, nivolumab, demonstrated encouraging response rates (33%), median event-free, and overall survival, compared with a historical cohort of contemporary patients treated with azacitidine-based therapies, with an acceptable safety profile. Biomarkers of response are yet to be determined. In this study, we leveraged a multiplexed immune assay to assess the functional states of CD4+ and CD8+ cells at a single-cell level in pretherapy bone marrows in 16 patients with relapsed/refractory AML treated with azacitidine/nivolumab. Effector CD4+ but not CD8+ cells had distinct polyfunctional groups and were associated with responses and better outcomes. Further evaluation of the polyfunctional strength index composition across cell types revealed that interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were the major drivers of enhanced polyfunctionality index of pretherapy CD4+ subset, whereas Granzyme B, IFN-γ, MIP-1b, and TNF-α drove the nonsignificantly enhanced pretreatment Polyfunctional Strength Index of CD8+ subset in the responders. Single-cell polyfunctional assays were predictive of response in AML and may have a potential role as a biomarker in the wider sphere of immunotherapy.