Successful bone tissue engineering requires the understanding of cellular activity in three-dimensional (3D) architectures and how it compares to two-dimensional (2D) architecture. We developed a perfusion culture system that utilizes fluid flow to mechanically load a cell-seeded 3D scaffold. This study compared the gene expression of osteoblastic cells in 2D and 3D cultures, and the effects of mechanical loading on gene expression in 2D and 3D cultures. MC3T3-E1 osteoblastlike cells were seeded onto 2D glass slides and 3D calcium phosphate scaffolds and cultured statically or mechanically loaded with fluid flow. Gene expression of OPN and FGF-2 was upregulated at 24 h and 48 h in 3D compared with 2D static cultures, while collagen 1 gene expression was downregulated. In addition, while flow increased OPN in 2D culture at 48 h, it decreased both OPN and FGF-2 in 3D culture. In conclusion, gene expression is different between 2D and 3D osteoblast cultures under static conditions. Additionally, osteoblasts respond to shear stress differently in 2D and 3D cultures. Our results highlight the importance of 3D mechanotransduction studies for bone tissue engineering applications.