Emerging evidence suggests that acute myeloid leukemia (AML) remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment, while suppressing normal hematopoiesis. 1 The influence of AML on bone tissue architecture and osteogenic cell differentiation has been documented in murine models, however, the impact of patient-derived AML cells on human BM stromal cells (BMSC) has only been investigated using conventional in vitro approaches. We assessed the differentiation potential of AML-derived BMSC using two in vivo models that recapitulate the complex organization of the human hematopoietic niche. We found that BMSC derived from pediatric AML patients: i) exhibit a reduced mature bone formation, ii) develop an osteoprogenitor-rich niche, iii) generate bone/BM organoids with a higher adipocytic differentiation, and iiii) support the formation of osteoclasts in a similar proportion to normal donor controls. All these aspects may contribute to the inhibition of normal hematopoietic stem and progenitor cell development and propagate selective blast cell survival and expansion. AML is a heterogeneous disorder characterized by the clonal proliferation of blasts in the BM. Leukemic cells compete with normal hematopoietic stem cells for niche occupation and this results in alterations of the BM microenvironment and the generation of a “leukemic niche” that selectively supports the malignant clone. 1 AML-induced changes in the BM microenvironment have been confirmed in multiple in vitro and in vivo studies. Murine AML models have shown several alterations in the BM niche components (eg, osteo-progenitors and osteoblasts) positively correlated with leukemogenesis. 2, 3 Similarly, a decrease in osteoblast number has been observed in the BM of AML patients together with reduced osteocalcin serum levels. 4 Moreover, studies have also reported that BMSC, one of the main cellular components of the hematopoietic niche, derived from AML patients exhibit a number of molecular and functional alterations, such as translocations, gene expression modifications, reduced clonogenic potential, decreased proliferation, higher senescence, impaired in vitro adipogenic and osteogenic differentiation, increased support of leukemia growth, and imbalanced regulation of endogenous hematopoiesis. 5-7 By contrast, other studies have reported that AML-BMSC display normal morphology and differentiation properties. 8 The humanized models are currently the only experimental system to reproduce an in vivo three-dimensional structure of the human BM niche, despite the limitations related to the potential interference of other components of recipient origins. 9 Several in vivo models have been described using normal or genetically modified human BMSC to generate a humanized BM niche that enables robust AML cell engraftment and allows evaluation of factors critical for the development/progression of leukemic cells within the niche. 9, 10 The aim of our work was to evaluate if AML-BMSC have undergone significant changes in their capability to form bone and a BM niche after exposure to patient leukemia in the BM. We isolated and expanded in vitro BMSC from the BM of newly diagnosed pediatric AML patients (AML-BMSC) and healthy donors (HD-BMSC). Patients and healthy controls were age-matched. BMSC donor characteristics are summarized in the Online Supplementary Table S1.