We are starting to work with mesenchymal stromal cells and are having a very difficult time differentiating them down the osteogenic lineage. We purchased bone marrow and amniotic membrane derived mesenchymal stromal cells from the company sciencellonline as well as their culturing reagents and osteogenic differentiation media. For the bone marrow stromal cells, we thawed them into a t75 flask coated with poly-lysine as per sciencell's protocol at passage 2. The cells grew a little faster than we thought they would and we ended up passing them at 100% confluence into 6-well plates at 10,000 cells per cm2. After 2 days they were confluent and we added the osteogenic differentiation media every 3-4 days. After 21 days though, no mineralization was seen. We though it might be due to the overconfluency when passing, but when we tried differentiating the amniotic membrane stromal cells that were passed at 70% confluency to 6-well plates and still saw no mineralization after 21 days in differentiation media, we are at a loss. Any suggestions are greatly appreciated.