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Acute myeloid leukemia (AML) results from a genetic alteration in hematopoietic stem cells (HSCs). Recent treatment techniques focused on the utilization of induced pluripotent stem cells (iPSC) as an avenue to introduce HSCs in patients with AML. In this study, we analyze whether OP9 cells assist in HSC differentiation through direct cell signaling or indirect cell signaling of iPSCs. We measured the expression levels of relevant cytokines known to play a role in hematopoiesis. Our data showed a significant upregulation of all the proteins we analyzed in the iPSC/OP9 direct coculture and significant downregulation in the iPSC/OP9 indirect coculture. Our results indicate that direct cell to cell contact with OP9 cells may be necessary to induce differentiation of iPSCs.