The aim of the current study was to prospectively assess the value of transesophageal ultrasound-guided fine needle aspiration (EUS-FNA) in the mediastinal staging of patients with non-small cell lung cancer (NSCLC) and CT negative for lymph node (LN) metastases, candidates for surgical resection. EUS-FNA was performed using the standard technique and LNs with at least one morphological feature suggestive of malignancy were sampled. Pathological exam of surgical specimens or tumor positive cytology was used as gold standard. Forty seven patients were included, 21% of whom had advanced disease (pN2) undetected by CT. EUS-FNA demonstrated LN metastases in 50% of them (11% of the whole series), and there were no false positives, resulting in a sensitivity, specificity, positive and negative predictive values and accuracy of 50%, 100%, 100%, 88% and 89%, respectively. In conclusion, EUS-FNA improves mediastinal staging in patients with NSCLC and CT negative for mediastinal nodes. Therefore, EUS-FNA should be considered in any patient with NSCLC and no distant metastases before any therapeutic decision is taken.