TDP-43 aggregation in the cytosol is a hallmark of neurodegenerative diseases like ALS. This study reveals that TDP-43 aggregation requires two key events: first, a high concentration of TDP-43 within stress granules, and second, oxidative stress. These events trigger intra-condensate demixing, leading to the formation of a TDP-43 enriched phase within stress granules, which then transitions into pathological aggregates. Mechanistically, intra-condensate demixing is driven by the unfolding of the RRM1 domain for disulfide bond formation and increased hydrophobic interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, the study successfully eliminates pathological TDP-43 aggregates in cells. The findings suggest that intra-condensate demixing is a critical intermediate step in the aggregation process, and that the combination of increased concentration within condensates and exposure to environmental stress could be a general pathway for protein aggregation. The study also shows that hydrophobic patch interactions and disulfide bond formation promote homotypic TDP-43 interactions that govern intra-condensate demixing. Additionally, the study demonstrates that the α-helix-to-β-sheet transition of the hydrophobic patch region is required for aggregation in the demixed TDP-43 phase. Finally, the study shows that oxidation-resistant TDP-43 variants with reduced self-assembly propensity abrogate pathological demixing in vivo. The results highlight the role of stress granules in TDP-43 aggregation and suggest that intra-condensate demixing is a key mechanism in the formation of pathological TDP-43 aggregates.TDP-43 aggregation in the cytosol is a hallmark of neurodegenerative diseases like ALS. This study reveals that TDP-43 aggregation requires two key events: first, a high concentration of TDP-43 within stress granules, and second, oxidative stress. These events trigger intra-condensate demixing, leading to the formation of a TDP-43 enriched phase within stress granules, which then transitions into pathological aggregates. Mechanistically, intra-condensate demixing is driven by the unfolding of the RRM1 domain for disulfide bond formation and increased hydrophobic interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, the study successfully eliminates pathological TDP-43 aggregates in cells. The findings suggest that intra-condensate demixing is a critical intermediate step in the aggregation process, and that the combination of increased concentration within condensates and exposure to environmental stress could be a general pathway for protein aggregation. The study also shows that hydrophobic patch interactions and disulfide bond formation promote homotypic TDP-43 interactions that govern intra-condensate demixing. Additionally, the study demonstrates that the α-helix-to-β-sheet transition of the hydrophobic patch region is required for aggregation in the demixed TDP-43 phase. Finally, the study shows that oxidation-resistant TDP-43 variants with reduced self-assembly propensity abrogate pathological demixing in vivo. The results highlight the role of stress granules in TDP-43 aggregation and suggest that intra-condensate demixing is a key mechanism in the formation of pathological TDP-43 aggregates.