Abstract Introduction: It is well established that the bone marrow stromal niche can serve as a protective environment in hematological malignancies such as AML by multiple cell contact-dependent and independent mechanisms. Intensive research of the bidirectional interactions between leukemic cells and mesenchymal stromal cells already highlighted numerous modes-of-action how malignant cells are capable of hijacking or altering their surroundings to their own favor. However, the entirety of underlying mechanisms is still incompletely understood. We found two small intracellular calcium-sensing molecules, S100A8 and S100A9, among the top upregulated genes in primary AML cells upon stromal contact. S100A8/A9 are members of the S100 protein family that, by functioning both as intracellular Ca2+ sensors and as extracellular mediators, can modulate cellular responses such as proliferation, migration, inflammation, and differentiation. Dysregulation of S100 protein expression is described as a common feature in several human cancers. Specifically in AML expression of S100A8 in leukemic cells predicts poor survival in de novo AML patients. Thus, we aimed to elucidate the underlying mechanisms of stroma-mediated S100A8/A9 upregulation as well as the consequences, and characterized S100A8/A9high AML cells in comparison to their S100A8/A9low counterparts in terms of gene expression pattern, differentiation, metabolic profiles, and chemoresistance. Methods: We co-cultured both AML cell lines and primary AML blasts in a contact-dependent and -independent manner with human bone marrow stromal cells. After co-culture AML cells were re-purified and analyzed by RNA sequencing, flow cytometry and quantitative real-time PCR. In some experiments, AML cells were sorted based on their S100A8/A9 protein levels and S100A8/A9high cells were compared to S100A8/A9low cells for their transcriptome. Results: We found S100A8 and S100A9 among the top upregulated genes in an unbiased transcriptome analysis of primary AML cells cultured in the presence of HS-5 cells compared tothe controls. Upregulation of S100A8/A9 could be confirmed in AML cell lines and was shown to be reversible. We could demonstrate that S100A8/A9 upregulation is mediated by soluble factors as cell-to-cell contact was not necessary and exosome-free conditioned medium from HS-5 cells did not induce S100A8/A9 gene expression. We found the Jak/STAT3 signaling being one major responsible pathway. The S100A8/A9high population was characterized by increased surface levels of maturation markers (such as CD14 and CD11b) as well as altered metabolically important transporters (e.g. for glucose, fatty and amino acids). Finally, we could demonstrate an increased chemoresistance of the S100A8/A9high cells. Conclusion: We could demonstrate bone marrow stroma-induced S100A8/A9 upregulation in AML cells is mediated by soluble factors activating the Jak/STAT3 pathway. S100A8/A9 leads to metabolic alterations and increased differentiation of AML cells conferring enhanced chemoresistance and thus represents a potential therapeutic target against AML. Disclosures No relevant conflicts of interest to declare.