Abstract Background: Leukemic and mesenchymal stem cells interact in the leukemic microenvironment and affect each other differently. This interplay has also important implications for the hematopoietic stem cell (HSC) biology and function. This study evaluated human HSC self-renewal potential and quiescence in an in vitro leukemic niche without leukemic cells. Methods: A leukemic niche was established by co-culturing mesenchymal stem cells with a fresh conditioned medium obtained from a leukemic (REH) cell line. After 3 days, the REH-conditioned medium was removed and freshly isolated CD34+ at a density of up to 100,000 cells/ml were added to the leukemic niche. CD34+ cell evaluaciones (cell cycle, self-renewal gene expression and migration capacity) were performed after 3 further days of coculture. Additionally, we preliminary investigated the soluble factors present in the leukemic niche and their effect on the mesenchymal stem cells. Statistical significance was assessed by Student¿s t test or the nonparametric test Kolmogorov¿Smirnov. Results: By co-culturing normal mesenchymal stem cells with the REH-conditioned medium we showed that hematopoietic stem cells, normally in a quiescent state, enter cell cycle and proliferate. This loss of quiescence was accompanied by an increased expression of Ki-67 and c-Myc, two well-known cell proliferation-associated markers. Two central regulators of quiescence GATA2 and p53 were also down regulated. Importantly, two genes involved in HSC self-renewal, Klf4 and the histone¿lysine N-methyltransferase enzyme Ezh2, were severely affected. On the contrary, c-Kit expression, the stem cell factor receptor, was upregulated in hematopoietic stem cells when compared to the normal niche. Interestingly, mesenchymal stem cells incubated with the REH-conditioned médium stopped growing, showed a flattened morphology with the appearance of small vacuoles, and importantly, became positive for the senescence-associated beta-galactosidase activity. Evaluation of the leukemic-conditioned médium showed increased IL-6 and IL-8, suggesting that these cytokines could be responsible for the observed changes. Conclusions: Our results showed that quiescence and self-renewal are severely affected in this leukemic niche. Thisin vitro leukemic niche, established without leukemic cells, will facilitate HSC gene expression evaluation and the development of therapeutic agents aimed to neutralize soluble factors and the cell signaling pathways involved in HSC alterations.