Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase. This study supports continued investigation in the maintenance of the mitochondrial DNA genome and its importance in stem cell maintenance.