Background: We have previously reported on characterising a novel mouse model of human Osteosarcoma (OS) based on the expression of a p53 shRNA transgene (p53.1224) in bone cells1 . Interestingly, endogenous p53 transcripts were detected in OS tumours but were not found to be functional. In this study, we investigate putative mechanisms of its inactivation. Methods: Mice expressing a p53 shRNA transgene (p53.1224) from a tetracycline-regulated promoter were bred with Osterix-Cre transgenic mice. The expression of tetracycline-regulated transactivator (tTA), Cre-GFP and p53.1224 shRNA were therefore restricted to the osteoblastic-lineage. OS tumour-derived cell lines were treated with pharmacological antagonists or siRNA against p53 inhibitors. Results: Twist1 (a known inactivator of p53) is highly expressed in shRNA-OS whole tumours and tumour-derived cell lines. However, the expression of p53-downstream genes was not elevated in Twist1 siRNA-treated OS cells. Furthermore, a lack of synergy was seen in p21 levels from OS cells treated with Doxorubicin and Nutlin3 (a Mdm2 antagonist). Conclusion: The p53 gene in the shRNA OS model is not inactivated via the Twist1 or Mdm2 pathway. Although it appears to be of a “wildtype” status, future experiments will focus on elucidating the mechanism of p53 inactivation in the shRNA OS model.