To address the involvement of PTEN/Akt signaling in DNA repair and genomic stability, we developed a shRNA-mediated PTEN knockdown cell line from HEK293T cells and evaluated its response to etoposide by analyzing γH2AX and Rad51 foci formation, cell cycle analysis, and chromosome damage. HEK PTEN knockdown cells were impaired in DNA repair associated with loss of G2/M checkpoint and reduced Rad51 foci formation. Furthermore, inhibition of Akt did not restore etoposide-induced G2/M arrest in PTEN knockdown cells, suggesting that loss of G2/M checkpoint in PTEN knockdown cells is Akt-independent. On the other hand, these cells become sensitive to etoposide when Akt was inhibited. Thus, loss of G2/M checkpoint and reduction of Rad51-mediated homologous recombination is responsible for the genomic instability of PTEN knockdown cells where activated Akt additionally contribute to strong survival signal. © 2012 Wiley Periodicals, Inc.