The study investigates the role of stress granules in the aggregation of the nuclear protein TDP-43, which is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The authors demonstrate that TDP-43 aggregation requires two key events: an up-concentration of TDP-43 in stress granules beyond a threshold and oxidative stress. These events collectively induce intra-condensate demixing, leading to a dynamic TDP-43-enriched phase within stress granules that subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is driven by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, the authors successfully eliminate pathological TDP-43 aggregates in cells. The findings suggest that stress granules can act as crucibles triggering TDP-43 aggregation via a key intra-condensate demixing process, and that the combination of increased concentration within condensates and simultaneous exposure to environmental stress could be a general mechanism for de novo protein aggregation in cells.The study investigates the role of stress granules in the aggregation of the nuclear protein TDP-43, which is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The authors demonstrate that TDP-43 aggregation requires two key events: an up-concentration of TDP-43 in stress granules beyond a threshold and oxidative stress. These events collectively induce intra-condensate demixing, leading to a dynamic TDP-43-enriched phase within stress granules that subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is driven by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, the authors successfully eliminate pathological TDP-43 aggregates in cells. The findings suggest that stress granules can act as crucibles triggering TDP-43 aggregation via a key intra-condensate demixing process, and that the combination of increased concentration within condensates and simultaneous exposure to environmental stress could be a general mechanism for de novo protein aggregation in cells.