Bone is the second most transplanted tissue after blood and engineered bone substitutes represent an attractive alternative to autologous bone grafts. Therefore, strategies are needed to facilitate the development of engineered bone graft substitutes in vitro. Perfusion bioreactors have been investigated as a component of engineered bone strategies because they supply oxygen and nutrients to cells seeded within a scaffold while applying a mechanical stimulus to the cells. Evidence with planar cell cultures has shown that dynamic flow regimens elicit an enhanced cellular response over steady flow regimens but dynamic perfusion strategies have not yet been translated to 3D scaffold architectures. The objective of this research is to show that pulsatile flow patterns will enhance osteogenic differentiation of bone marrow stromal cells seeded in porous scaffolds over a continuous flow pattern as indicated by levels of alkaline phosphatase activity and the bone extracellular matrix protein, osteopontin. Our results indicate that all flow conditions enhanced alkaline phosphatase activity and osteopontin expression. Additionally, these markers are preferentially enhanced by pulsatile flow over continuous flow. A trend of increasing alkaline phosphatase activity with decreasing pulse frequency suggests that cells are sensitive to frequency of pulsatile flow. These results indicate that dynamic perfusion may be a useful component of the engineered bone tissue strategy.