Abstract At homeostasis, type 3 innate lymphoid cells (ILC3s) are tissue-resident and have key roles in maintaining mucosal immunity. Recently, the expansion of ILC3s has been described in solid tumors; however, the mechanism driving this finding is not completely understood. In the setting of mouse and human acute myeloid leukemia (AML), we have observed an expansion of ILC3s. Using an AML murine model, we observed a 7-fold increase in ILC3s in the intestine (the normal ILC3 niche) of leukemic mice relative to controls and a 3-fold increase of ILC3s in the bone marrow (intestine: 0.208% +/− 0.04 AML vs 0.032% +/− 0.04 WT, n=5, p<0.001; bone marrow: 1.312% +/− 0.20 AML vs 0.067% +/− 0.20 WT, n=5, p<0.0001). We hypothesized that the ILC3 increase was due at least in part to increased ILC3 differentiation. To address this hypothesis, we isolated the innate lymphoid cell precursor (ILCP) from normal human donors and co-cultured with AML cells which resulted in increased ILC3 frequency (59% +/− 5% with AML vs 28% +/− 5% without AML, n=15, p <0.001). This observation was also recapitulated in a human PDX model in NSG mice (3.22% +/− 7.8% without AML vs 23.91% +/− 7.8% with AML, n=10; p<0.0001). Our published data have shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. By manipulating AHR activity ex vivo, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (60% +/− 5.5% with AML vs 27% +/− 5% AML+AHR inhibitor, n=15, p<0.001). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion by skewing ILCP differentiation towards ILC3. Future studies will define the role of ILC3s in AML progression. Supported by grant from NIH (1R01CA255860-01A1)