ysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21 and p16INK4a-Rb-dependent DNA damage responses (DDR). The p16INK4a tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence and limits stem cell function /in vivo/. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16INK4a in response to telomere dysfunction remains unclear. Here, we generated /Pot1b∆/∆, p16-/-/ mice to address the function of p16INK4a in the setting of telomere dysfunction /in vivo/. Surprisingly, we found that deletion of /p16INK4a/significantly accelerated organ impairment, with functional defects in highly proliferative organs, including the hematopoietic system, small intestine and testes. ATR-dependent DDR is robustly activated in /Pot1b∆/∆; p16-/-/hematopoietic cells, resulting in p53 stabilization, increased p21-dependent cell cycle arrest and elevated p53-dependent apoptosis. Our results provide the first experimental evidence that dysfunctional telomeres cooperate with /p16INK4a/ deficiency to robustly activate an ATR-dependent DDR in hematopoietic cells, resulting in bone marrow failure and premature death.