Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter-enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.