This study evaluates the use of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) to predict response to neoadjuvant chemotherapy (neoCTx). PDOs were generated from biopsies of 120 patients with locally advanced EGC. The PDOs were tested for their response to single chemotherapeutics and the FLOT regimen (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel). The response of PDOs was correlated with the patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n=13) and confirmed in an independent validation cohort (n=13). EGC PDOs showed diverse responses to single chemotherapeutics and the FLOT 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; τ=0.694, P=2.541e-08). FLOT testing showed high diagnostic precision with an AUC of 0.994. The discriminative ability of 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 vitro drug testing of EGC PDOs has high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. The high rate of successful PDO expansion from biopsies supports the definition of a threshold that allows treatment stratification, paving the way for interventional trials exploring PDO-guided treatment of EGC patients. The study demonstrates that PDOs can be used to predict treatment response in EGC patients, which could lead to more personalized treatment strategies. The results suggest that PDOs have the potential to improve patient outcomes by enabling the selection of the most effective treatment for individual patients.This study evaluates the use of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) to predict response to neoadjuvant chemotherapy (neoCTx). PDOs were generated from biopsies of 120 patients with locally advanced EGC. The PDOs were tested for their response to single chemotherapeutics and the FLOT regimen (5-fluorouracil, leucovorin, oxaliplatin, and docetaxel). The response of PDOs was correlated with the patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n=13) and confirmed in an independent validation cohort (n=13). EGC PDOs showed diverse responses to single chemotherapeutics and the FLOT 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; τ=0.694, P=2.541e-08). FLOT testing showed high diagnostic precision with an AUC of 0.994. The discriminative ability of 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 vitro drug testing of EGC PDOs has high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. The high rate of successful PDO expansion from biopsies supports the definition of a threshold that allows treatment stratification, paving the way for interventional trials exploring PDO-guided treatment of EGC patients. The study demonstrates that PDOs can be used to predict treatment response in EGC patients, which could lead to more personalized treatment strategies. The results suggest that PDOs have the potential to improve patient outcomes by enabling the selection of the most effective treatment for individual patients.