Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a component of the multi-aminoacyl-tRNA synthetase complex and is involved in diverse cellular processes. Given that AIMP3 deficiency causes early embryonic lethality in mice, AIMP3 is expected to play a critical role in early mouse development. To elucidate a functional role of AIMP3 in early mouse development, we induced AIMP3 depletion in mouse embryonic stem cells (mESCs) derived from blastocysts of AIMP3f/f; CreERT2 mice. In the present study, AIMP3 depletion resulted in loss of self-renewal and ability to differentiate to three germ layers in mESCs. AIMP3 depletion led to accumulation of DNA damage by blocking double-strand break repair, in particular homologous recombination. Through microarray analysis, the p53 signaling pathway was identified as being activated in AIMP3-depleted mESCs. Knockdown of p53 rescued loss of stem cell characteristics by AIMP3 depletion in mESCs. These results imply that AIMP3 depletion in mESCs leads to accumulation of DNA damage and p53 transactivation, resulting in loss of stemness. We propose that AIMP3 is involved in maintenance of genome stability and stemness in mESCs.