The bone marrow (BM) stroma represents a protective niche for acute myeloid leukemia (AML) cells. However, the complex underlying mechanisms remain to be fully elucidated. We found 2 small, intracellular, calcium-sensing molecules, S100A8 and S100A9, among the top genes being upregulated in primary AML blasts upon stromal contact. As members of the S100 protein family, they can modulate such cellular processes as proliferation, migration, and differentiation. Dysregulation of S100 proteins is described as a predictor of poor survival in different human cancers, including increased S100A8 expression in de novo AML. Thus, we wanted to decipher the underlying pathways of stroma-mediated S100A8/A9 induction, as well as its functional consequences. Upregulation of S100A8/A9 after stromal cross talk was validated in AML cell lines, was contact independent and reversible and resulted in accumulation of S100A8/A9high cells. Accordingly, frequency of S100A8/A9high AML blasts was higher in the patients' BM than in peripheral blood. The S100A8/A9high AML cell population displayed enhanced utilization of free fatty acids, features of a more mature myeloid phenotype, and increased resilience toward chemotherapeutics and BCL2 inhibition. We identified stromal cell-derived interleukin-6 (IL-6) as the trigger for a Jak/STAT3 signaling-mediated S100A8/A9 induction. Interfering with fatty acid uptake and the IL-6-Jak/STAT3 pathway antagonized formation of S100A8/A9high cells and therapeutic resistance, which could have therapeutic implications as a strategy to interfere with the AML-niche dynamics.