The early preimplantation embryo is susceptible to a range of exogenous stresses which result in their reduced long-term developmental potential. The P53 tumour suppressor protein is normally held at low levels in the preimplantation embryo and we show that culture stress induces the expression of a range of canonical P53-response genes (Mdm2, Bax and Cdkn1a). Culture stress caused a P53-dependent loss of cells from resulting blastocysts, and this was most evident within the inner cell mass population. Culture stress increased the proportion of cells expressing active caspase-3 and undergoing apoptosis, while inhibition of caspase-3 increased the number of cells within the inner cell mass. The P53-dependent loss of cells from the inner cell mass was accompanied by a loss of NANOG-positive epiblast progenitors. Pharmacological activation of P53 by the MDM2 inhibitor, Nutlin-3, also caused increased P53-dependent transcription and the loss of cells from the inner cell mass. This loss of cells could be ameliorated by simultaneous treatment with the P53 inhibitor, Pifithrin-α. Culture stress causes reduced signalling via the phosphatidylinositol-3-kinase signalling pathway, and blocking this pathway caused P53-dependent loss of cells from the inner cell mass. These results point to P53 acting to limit the accumulation and survival of cells within the pluripotent lineage of the blastocyst and provide a molecular framework for the further investigation of the factors determining the effects of stressors on the embryo's developmental potential.