Background: Diagnosis of a foregut duplication cyst is of great clinical impact. A definitive diagnosis of a foregut duplication cyst can avert the need for major thoracic surgery in the otherwise asymptomatic individual. This study sought to evaluate the safety and the utility of EUS and EUS-guided FNA (EUSFNA) in the diagnosis of foregut duplication cysts. Methods: Over a period of 4 years, 4771 patients underwent EUS for various indications at two EUS referral centers. EUS findings were consistent with a mediastinal cyst in 30 cases. EUSFNA was performed in 22 patients. A definitive diagnosis was established based on cytology, surgical pathology, and/or clinical follow-up. FNA was done with 22- gauge needles and antibiotic prophylaxis. Results: The appearance of cyst contents on EUS ranged from completely anechoic (23 cases) to hypoechoic (7 cases). Hypoechoic cystic lesions contained echogenic foci. All anechoic lesions were confirmed as benign duplication cysts based on cytology, pathology, and clinical follow up. Hypoechoic cystic lesionswere confirmed to be benign duplication cysts in 4 cases. Three cases proved to bemalignant or granulomatous necrotizing lymph nodes. No periprocedural complications occurred. Conclusions: Variation exists in the EUS appearance of benign mediastinal cysts. EUS-FNA of mediastinal cysts with smaller-gauge needles, and antibiotic prophylaxis appears safe and can provide a definitive diagnosis in atypical mediastinal cystic lesions. Background: Diagnosis of a foregut duplication cyst is of great clinical impact. A definitive diagnosis of a foregut duplication cyst can avert the need for major thoracic surgery in the otherwise asymptomatic individual. This study sought to evaluate the safety and the utility of EUS and EUS -guided FNA (EUSFNA) in the diagnosis of foregut duplication cysts. Methods: Over a period of 4 years, 4771 patients underwent EUS for various indications at two EUS referral centers. EUS findings were consistent with a mediastinal cyst in 30 cases. EUSFNA was performed in 22 patients. A definitive diagnosis was established based on cytology, surgical pathology, and / or clinical follow-up. FNA was done with 22-gauge needles and antibiotic prophylaxis. Results: The appearance of cyst contents on EUS ranged from completely anechoic (23 cases) to hypoechoic (7 cases). Hypoechoic cystic lesions contained echogenic foci. All anechoic lesions were confirmed as benign duplication cysts based on cytology, Pathology, and clinical follow-up. Hypoechoic cystic lesionswere confirmed to be benign duplication cysts in 4 cases. Three cases proved to bemalignant or granulomatous necrotizing lymph nodes. No periprocedural causes occurred. Conclusions: Variation exists in the EUS appearance of benign mediastinal cysts. EUS-FNA of mediastinal cysts with smaller-gauge needles, and antibiotic prophylaxis appeared safe and can provide a definitive diagnosis in atypical mediastinal cystic lesions.