Defective pluripotent cells are removed from embryos prior to differentiation, presumably due to upregulation of the p53 pathway. However, the mechanism underlying p53 protein activation is still unknown. Embryonic stem cells (ESCs), corresponding to cells of the preimplantation blastocyst, likely have similar mechanisms for abnormal cell elimination. Using a mouse ESC cell line with inducible ulk1 gene expression, we showed that Ulk1 upregulation is accompanied by p53 phosphorylation on Ser15. ESCs tolerated the activated p53 and did not undergo apoptosis or cell cycle blockade upon Ulk1 overexpression. However, massive cell death was observed after retinoic acid treatment, suggesting a role of Ulk1-induced p53 activation in the elimination of defective pluripotent cells prior to differentiation.