Because the microenvironment that supports hematopoietic stem cell (HSC) proliferation and differentiation is not fully understood, we adapted a heterotopic bone formation model as a new approach for studying the HSC microenvironment in vivo. Endogenous HSCs homed to tissue-engineered ossicles and individually sorted HSCs from ossicles were able to reconstitute lethally irradiated mice. To further explore this model as a system to study the stem cell niche, ossicles were established with or without anabolic parathyroid hormone (PTH) treatment during the 4-week course of bone development. Histology and micro–computed tomography showed higher bone area-to-total area ratios, thicker cortical bone and trabecular bone, significantly higher bone mineral density and bone volume fraction in PTH-treated groups than in controls. By an in vivo competitive long-term reconstitution assay, HSC frequency in the ossicle marrow was 3 times greater in PTH groups than in controls. When whole bone marrow cells were directly injected into the ossicles after lethal irradiation, the PTH-treated groups showed an enhanced reconstitution rate compared with controls. These findings suggest the residence of HSCs in heterotopic bone marrow and support the future use of this ossicle model in elucidating the composition and regulation of the HSC niche.