Green fluorescent protein (GFP) expression is widely used for tracking of cells in vivo. Three human osteosarcoma cell lines were transfected with GFP and analyzed for expression of tissue markers and physiology. GFP-mediated increased tumor aggression in vivo in one cell line was corroborated by altered expression of bone markers and osteoblast-like cell biology in vitro as a result of transfection. The use of GFP for cell tracking in osteosarcoma animal models should therefore be used with caution.