Background: Despite normal mediastinal (N2) lymph nodes shown on positron emission tomography (PET) and CT, some physicians routinely perform mediastinoscopy and/or endoscopic ultrasound fine-needle aspiration (EUS-FNA) in patients with non-small cell lung cancer (NSCLC). Methods: A prospective trial on patients with NSCLC who were clinically staged N2 negative by both integrated PET/CT and CT scan. All underwent mediastinoscopy and EUS-FNA and if N2 negative underwent thoracotomy with thoracic lymphadenectomy. Results: There were 153 patients (107 men). Of these, 136 patients were clinically staged N0 and 17 patients were clinically staged N1. Of the 136 patients who were staged as N0, 5 patients (3.7%) had positive EUS-FNA results (three in the subcarinal node), and 4 patients (2.9%) had positive mediastinoscopy results (all in the #4R node; one was N3). Six of the remaining 127 patients (4.7%) had N2 disease after resection. Seventeen patients were clinically staged as N1 by integrated PET/CT. Four patients (23.5%) had positive EUS-FNA results (two in the subcarinal node), 3 patients (17.6%) had positive mediastinoscopy results (all in #4R node; two were N2 and one was N3), and none of the remaining 10 patients had N2 disease after resection. Patients with unsuspected N2 disease were twice as likely (relative risk, 2.1; 95% confidence interval, 1.24 to 2.51; p = 0.02) to have a maximum standardized uptake value (maxSUV) > 10 and poorly differentiated cancer (relative risk, 2.1; 95% confidence interval, 1.14 to 2.38; p = 0.03). Conclusion: We do not recommend routine mediastinoscopy or EUS-FNA in patients who are clinically staged as N0 after both integrated PET/CT and CT. However, these procedures should both be considered in patients clinically staged as N1 after PET/CT, and/or in those with adenocarcinoma, upper-lobe tumors, or tumors with a maxSUV ≥ 10.