Real-time endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an established technique for invasive mediastinal staging of non-small cell lung cancer (NSCLC). Needle-based techniques are now recommended as a first-line diagnostic modality for mediastinal staging. Accurate performance of systematic staging with EBUS-TBNA requires a detailed knowledge of mediastinal anatomy. This examination begins at the N3 lymph nodes, progressing through the N2 and N1 lymph node stations, unless a higher station lymph node is positive for malignant cells by rapid on-site cytologic examination. Objective methods of identifying EBUS-TBNA targets include sampling any lymph node station with a visible lymph node or with a lymph node greater than 5 mm in short axis. Three passes per station or the use of rapid on-site cytologic examination with identification of diagnostic material (tumor or lymphocytes) up to five passes are well-established techniques. Obtaining sufficient tissue for molecular profiling may require performing more than three passes. The operating characteristics of EBUS-TBNA are similar to mediastinoscopy. However, mediastinoscopy should be considered in the setting of a negative EBUS-TBNA and a high posterior probability of N2 or N3 involvement.