Apoptosis occurs primarily in the blastocyst inner cell mass (ICM), cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo derived blastocysts were compared to control embryos X-irradiated at the 2-cell stage and cultured to Day 5. Analysis of DNA by Comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured 2-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo, or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices which alter apoptosis in human embryos and/or select embryos for transfer which potentially lack a functional Trp53 gene need to be carefully considered.