This study investigates the use of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) to predict response to neoadjuvant chemotherapy (neoCTx). Endoscopic biopsies from 120 patients with locally advanced EGC were cultured to generate PDOs, which were then tested for their response to the FLOT regimen (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel). The PDOs' responses were correlated with the patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established and validated in two cohorts. The results showed that PDOs reflected diverse responses to single chemotherapeutics and the FLOT combination regimen. In the exploratory cohort, PDOs' response to 5-FU and FLOT correlated with the patients' pathological response (Kendall's τ = 0.411, P = 0.001; Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision was achieved with an AUC ROC of 0.994. The PDO-based FLOT testing allowed the definition of a threshold that classified FLOT responders from non-responders with high sensitivity (90%), specificity (100%), and accuracy (92%) in an independent validation cohort. The study concludes that in vitro drug testing of EGC PDOs has high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment, paving the way for interventional trials exploring PDO-guided treatment of EGC patients.This study investigates the use of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) to predict response to neoadjuvant chemotherapy (neoCTx). Endoscopic biopsies from 120 patients with locally advanced EGC were cultured to generate PDOs, which were then tested for their response to the FLOT regimen (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel). The PDOs' responses were correlated with the patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established and validated in two cohorts. The results showed that PDOs reflected diverse responses to single chemotherapeutics and the FLOT combination regimen. In the exploratory cohort, PDOs' response to 5-FU and FLOT correlated with the patients' pathological response (Kendall's τ = 0.411, P = 0.001; Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision was achieved with an AUC ROC of 0.994. The PDO-based FLOT testing allowed the definition of a threshold that classified FLOT responders from non-responders with high sensitivity (90%), specificity (100%), and accuracy (92%) in an independent validation cohort. The study concludes that in vitro drug testing of EGC PDOs has high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment, paving the way for interventional trials exploring PDO-guided treatment of EGC patients.