This study was designed to test the hypothesis that the mechanical properties of a trabecular bone substitute can be enhanced through in vitro tissue formation. Our specific objectives were to (1) determine the effects of in vitro marrow stromal cell-mediated tissue deposition upon a trabeculated hydroxyapatite scaffold on the strength and toughness of the resulting bone substitute; and (2) identify and characterize regions of newly deposited matrix and mineral. This work provides a basis for future investigations aimed at transforming a brittle hydroxyapatite scaffold into an osteoinductive, biomechanically functional implant through in vitro bone deposition. As hypothesized, the mechanical properties of the trabecular bone substitutes were significantly enhanced by in vitro tissue formation. As a result of cell seeding and a 5 week culture protocol, mean strength increased by 85% (p = 0.008) and energy to fracture increased by 130% (p = 0.003). Accompanying the enhancement of mechanical properties was the deposition of significant amounts of bone matrix and mineral. Fluorescence imaging, scanning electron microscopy, electron probe microanalysis, and nanoindentation confirmed the presence of bonelike mineral with Ca/P ratio, modulus, and hardness similar to that within human and rat trabecular bone tissue. This new mineralization was found to exist within a newly deposited parallel-fibered matrix both encasing and bridging between scaffold trabeculae. Taken as a whole, our results establish the feasibility of the production of an osteoinductive hydroxyapatite-based trabecular bone substitute with mechanical properties enhanced through in vitro bone deposition.