Ets-related transcription factor GA-binding protein alpha (GABPα), also known as NRF-2, is expressed in a variety of cell types and is involved in cellular functions such as cell cycle regulation, apoptosis, mitochondrial biogenesis and differentiation. Here we generated Gabpa conditional knockout embryonic stem (ES) cells and characterized its cellular phenotypes. Disruption of Gabpa revealed that the proliferation of Gabpa-null ES cells was drastically repressed and cells started to die within 2 days. The repressed proliferation of Gabpa-null ES cells was recovered by artificially forced expression of GABPα. Transcriptional profiles of Gabpa-null ES cells showed that approximately 30% of " G1 to S cell cycle control genes " were changed. Expression analysis showed that p53 mRNA levels were comparable; however, p53 target genes, including Cdkn1a/p21, Mdm2, and Gadd45a, were upregulated and cell cycle-related genes, including Cyclin D1/D2 and Cyclin E1/E2, were downregulated in Gabpa-null ES cells. G1 cell cycle arrest was induced by Gabpa disruption. Interestingly, p53 and cleaved Caspase3 expressions were enhanced in the cells and reduced proliferation as well as cell death of Gabpa-null ES cells were rescued by either transfection of p53 RNAi or treatment of the p53 inhibitor pifithrin-α. These results suggest that GABPα inhibits the accumulation of p53 and is involved in the proliferation and survival of ES cells.