Updated project metadata. In this study we characterize and investigate the effect of the loss of Ku70 protein in a conditional Ku70 knockout TREx-293 cell system. Loss of Ku70 protein levels was directly correlated with a loss of cell viability, supporting the observation that Ku performs an essential role in human cells. Interestingly, critical loss of average telomere length was not observed in Ku70 knockouts generated. Global quantitative proteomic analysis of whole cell extracts from Ku70 knockouts following depletion of Ku70 may indicate that Ku’s essential role in humans is not at telomeres, but a non-canonical role which is not yet fully understood.