To explore the effect of Trp53 and Rb1 deficiency in Ctsk‐expressing cells on activity and differentiation of osteoblasts, we isolated Ctsk+ cells from the cortical bone of osteosarcoma area11 and explored the function of Trp53 and Rb1 in these cells in vitro by performing alkaline phosphatase (ALP) staining, ALP activity assays, and alizarin red S (ARS) staining. This evidence concerns the gene TP53 and osteosarcoma.