The human bone marrow (hBM) is a complex organ critical for hematopoietic and immune homeostasis, and where many cancers metastasize. Understanding the fundamental biology of the hBM in health and diseases remain difficult due to complexity of studying or manipulating the BM in humans. Accurate biomaterial-based in vitro models of the hBM microenvironment are critical to further our understanding of the BM-niche and advancing new clinical interventions. Here we report a unique, 96-well format, microfluidic hBM-on-a-chip that incorporates the endosteal, central marrow, and perivascular niches of the human BM. Osteogenic differentiation of donor human mesenchymal stromal cells (MSCs) produced robust mineralization on the bottom surface ("bone-like endosteal layer") of the device, and subsequent seeding of human endothelial cells and MSCs in a fibrin-collagen hydrogel network ("central marrow") on the top created an interconnected 3D microvascular network ("perivascular niche"). The 96-well format allows eight independent "chips" to be studied in one plate, thereby increasing throughput and reproducibility. We show that this complex, multi-niche microtissue accurately mimics hBM composition and microphysiology, while providing key insights on hematopoietic progenitor dynamics. Presence of the endosteal niche decreased the proliferation and increased maintenance of CD34 hematopoietic stem cells (HSCs). Upon exposure to radiation, HSCs in the hBM-chips containing endosteal niches were less frequently apoptotic, suggesting a potentially radio-protective role of the osteoblast surface. Our methods and results provide a broad platform for creating complex, multi-niche, high-throughput microphysiological (MPS) systems. Specifically, this hBM-on-a-chip opens new opportunities in human bone marrow research and therapeutics development, and can be used to better understand normal and impaired hematopoiesis, and various hBM pathologies, including cancer and BM failures.