Exogenous (human or mouse) mesenchymal stem cells (MSCs) seed in HA scaffold and transplant subcutaneously in immunodeficient mice, the cells can finally form bone tissues in the in vivo environment. This protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs) in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by tissue engineering approaches in vivo, and it also fits for examining the roles of different factors in bone formation.