Abstract Body: Chemotherapeutic agents can disrupt stem cell pluripotency and self-renewal, yet how hiPSCs adapt to such stress remains unclear. Doxorubicin (Dox), a widely used cancer drug, is known for its cytotoxic effects at high doses; however, its impact at low doses on hiPSC proliferation and self-renewal remains poorly characterized. We hypothesize that low-dose Dox induces an adaptive response in hiPSCs, reducing cell size while preserving pluripotency and self-renewal. JHU001 hiPSCs were treated with 50 nM Dox for 2 hours (hiPSC-D50) and compared to vehicle-treated controls (hiPSC-D0). Both hiPSC-D50 and -D0 were cultured in E8 flex medium, passaged five times, and cryopreserved. Passage 5 was selected to assess stable adaptation following Dox withdrawal. To quantify cell area changes, a total of 161 (hiPSC-D50) and 140 (hiPSCS-D0) individual cells were manually segmented (n= 6) from phase-contrast images using ImageJ. hiPSC-D50 and -D0 cells were examined for pluripotent gene expression (Oct-4, Nanog, and SSEA-4) by immunocytochemistry using epifluorescence microscopy. hiPSC-D50 cells exhibited a significantly smaller mean area (80.96 ± 1.54 micrometer square) compared to hiPSC-D0 cells (141.63 ± 3.36 micrometer square), corresponding to a 1.75-fold reduction (p= 3.2e-46). These findings suggest that low-dose Dox induces a stable reduction in hiPSC size, supporting an adaptive Dox response. Furthermore, immunocytochemistry analysis revealed an upregulation of pluripotency markers, with a 1.3-fold (Oct4), 1.9-fold (Nanog), and 2.9-fold (SSEA-4) increase in hiPSC-D50 relative to -D0 (p= 0.00057, 0.018, 0.0000026, respectively). These results indicate that low-dose Dox reprograms the size threshold, allowing hiPSCs to maintain self-renewal at a reduced size. This aligns with the Sizer model, which states that cells must reach a critical size before division. Furthermore, sustained self-renewal despite size constraints suggests that cell cycle regulators accumulate rapidly, supporting the Accumulating Activator model, where a size-dependent activator triggers mitotic entry at a reduced size. Understanding the molecular mechanisms of size-dependent pluripotency and self-renewal may provide insights into optimizing hiPSC survival and regenerative therapies under chemotherapeutic stress, potentially informing strategies to mitigate toxicity in cancer patients.