Bone regenerative medicine via tissue engineering is expected to be an alternative treatment for conventional autogenous bone graft, as it is less invasive. One of the best triads for bone engineering is bone marrow stromal cells, calcium phosphate ceramics, and bone morphogenetic protein (BMP). However, the optimal mixing conditions for BMP-induced osteoblasts and ceramic granules remain unclear. Therefore, we investigated the effect of the mixing conditions for cell scaffolds on the bone-forming potential. The cells were mixed with beta-tricalcium phosphate (beta-TCP) granules followed by osteoblast induction with recombinant human BMP-2 (rhBMP-2) (first mixture), or were first induced with rhBMP-2 on plastic dishes and then mixed with the beta-TCP granules (last mixture) just prior to the operation. Both the first and last mixtures were transplanted into nude mice subcutaneously, with the amount of bone formation analyzed histomorphometrically. In addition, cell numbers and alkaline phosphatase (ALP) activity before transplantation was determined in both the mixtures. In vitro analyses revealed that cell numbers were greater in the last mixture, whereas ALP activity was greater in the first mixture. In vivo analyses revealed that the first mixture was much more osteogenic than the last mixture with respect to new bone formation and osteocalcin synthesis. These data suggest that cell-scaffold mixing conditions have a significant influence on the bone-forming capacity via bone engineering and that first mixture might be the optimal condition for rhBMP-2-induction of human osteoblasts.