AbstractMaintaining a definite and stable pool of dividing stem cells plays an important role in organ development. This process requires an appropriate progression of mitosis for proper spindle orientation and polarity to ensure the ability of stem cells to proliferate and differentiate correctly. Polo-like kinases (Plks)/Polo are the highly conserved serine/threonine kinases involved in the initiation of mitosis as well as in the progression of the cell cycle. Although numerous studies have investigated the mitotic defects upon loss of Plks/Polo in cells, little is known about the in vivo consequences of stem cells with abnormal Polo activity in the context of tissue and organism development. The current study aimed to investigate this question using theDrosophilaintestine, an organ dynamically maintained by the intestinal stem cells (ISCs). The results indicated that thepolodepletion caused a reduction in the gut size due to a gradual decrease in the number of functional ISCs. Interestingly, thepolo-deficient ISCs showed an extended G2/M phase and aneuploidy and were subsequently eliminated by premature differentiation into enterocytes (ECs). In contrast, the constitutively active Polo (poloT182D) suppressed ISC proliferation, induced abnormal accumulation of β-tubulin in cells, and drove ISC loss via apoptosis. Therefore, Polo activity should be properly maintained for optimal stem cell function. Further analysis suggested thatpolowas a direct target gene of Sox21a, a Sox transcription factor that critically regulates stem cell activity. Together, this study provided a novel perspective on the correlation between the progression of mitosis and the ISC function inDrosophila.