Abstract The interactions that occur between leukemic cells and the microenvironment are currently poorly understood. The definition of novel pathways that deliver survival and drug resistance signaling from the milieu to leukemic cells may lead to the discovery of new drugs that are able to target microenvironmental cues perpetuating the leukemic clones. Since the bone marrow (BM) environment is comprised of a complex assortment of different cell types, matrices and soluble and membrane-bound factors, it is crucial to start delineating specific cell-cell interactions and their effects on leukemic cell survival. Mesenchymal stem cells (MSC) are a fundamental component of the BM niche as they support hematopoiesis and are able to differentiate into neighboring cellular types. Therefore, in these studies we investigated the effect of Stro-1+ MSC on the growth and survival of KG1a, a primitive, differentiation-resistant subtype of acute myeloid leukemia. MSC and KG1a cells were co-cultured for 3 days under contact or non-contact conditions, at incremental ratios of 1:1, 1:5, 1:10 and 1:100 respectively, while maintaining a constant initial total number of cells for each dilution (n=3). In transwell non-contact cultures, KG1a cells proliferated at the same rate as KG1a alone with a 3 to 5 fold increase in cell numbers, regardless of the ratio. By contrast, when KG1a were cultured in direct contact with MSC, KG1a growth inhibition was directly proportional to the MSC:KG1a ratio. Specifically, MSC cultured with KG1a cells at ratios of 1:1, 1:5, 1:10, and 1:100 inhibited the growth of KG1a cells by 84%, 76%, 74%, and 45%, respectively while remaining 99-100% viable. Thus, we next performed cell cycle analysis using flow cytometry and found that while the cell cycle distribution of KG1a cultures were: 23% in G0/G1; 19% in G2/M; and 58% in S phase, KG1a co-cultured with MSC had undergone cell cycle arrest in S-phase (98%). Although our results contrast with prior studies showing that MSC inhibited KG1a in G1phase through the production of soluble factors, it is possible that the differences seen are due to the population of MSC used. Within the MSC, Stro-1+ cells constitute a primitive population of cells in intimate relation with the osteoblastic niche, the role of which is to retain stem cells in a quiescent state. Therefore, studies are currently underway to identify molecules responsible cell cycle arrest upon KG1a/MSC interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 538.