Abstract Hematopoiesis develops in the bone marrow (BM) where multiple interactions regulate the differentiation and preservation of hematopoietic stem and progenitor cells (HSPCs). Immune‐deficient murine models have enabled the analysis of molecular and cellular regulation of human HSPCs, but the physiology of these models is questioned as human hematopoietic cells develop in xenogenic microenvironments. In this study, we thoroughly characterized a humanized (h) in vivo BM model, developed from fetal (F/) and post‐natal (P‐N/) mesenchymal stromal cell (MSC) differentiation (called hOssicles [hOss]), in which human hematopoietic cells are generated following the transplantation of CD34+ cells. Serial isolation and transplant experiments of hMSCs and HSPCs from hOss revealed the dynamic nature of these hBM niches. hOss modified human hematopoietic development by modulating myeloid/lymphoid cell production and HSPC levels, with no major transcriptional changes in HSPCs at the single‐cell level. Clonal tracking using genetic barcodes highlighted hematopoietic cell cross‐talks between the endogenous murine BM and hOss and differences in clonal myeloid/multipotent cell production between F/hOss and P‐N/hOss, uncovering ontogeny‐related impact of the BM on human hematopoietic cell production.
