Updated project metadata. Telomeric repeat sequences are prone to induce replication stress and one of the core telomere binding proteins, TRF1, is thought to counteract these problems. The molecular mechanisms coming into play upon TRF1 loss were not clearly defined though. This study combines an inducible TRF1 loss with telomere-targeted proteomics to analyze the incurring changes. The results show that loss of TRF1 causes important alterations in telomere associated proteins and the new profile resembles in a number of ways that of cells in which telomeres are maintained by telomerase-independent mechanisms, named ALT. The presence of DNA repair proteins (MRN complex, the SMC5/6 complex), some increases in telomere colocalization with PML bodies, sister chromatid exchanges and clear signs of increased TERRA transcription and mitotic break-induced replication are part of these phenotypes. The data also show that these early ALT phenotypes can be differentiated from drug induced replication stress, and part of this ALT response is conserved in human cells. These studies therefore point to a critical role for TRF1 in preventing the emergence of ALT and hence immortalization of cells. Given that the details for how ALT actually is initiated remained unclear, these findings are an important step forward and will spur new research into that question.