Background: Although endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) using a 22-gauge needle has emerged as an accurate, minimally invasive, and safe technique for accessing undiagnosed mediastinal adenopathy, particularly in patients with lung cancer, the small sample size obtained by a conventional needle may limit the accuracy of the result. Objectives: We evaluated the safety and efficacy of obtaining specimens using 21-gauge needles, comparing diagnostic yield with conventional 22-gauge needles. Methods: Participants consisted of 56 consecutive patients with mediastinal lesions referred to our institution for diagnostic EBUS-TBNA. The 21-gauge needle group (21 G) included 24 patients with 6 nonmalignancies and the 22-gauge needle group (22G) included 32 patients with 9 nonmalignancies. Final diagnosis was based on cytology, histology, surgical results, appropriate clinical pictures on examination, and/or clinical follow-up. Results: Comparing 21G and 22G, inadequate material rates were 0% versus 3.1% in cytology, and 4.2% versus 18.8% in histology, respectively. Accuracy in cytology, histology, and combined cytology and/or histology were 91.7% versus 65.6% (P=0.02), 95.8% versus 81.3% (P=0.11), and 100% versus 84.4% (P=0.04), respectively. After limiting cases to suspected malignancies, sensitivity in cytology, histology, and combined cytology and/or histology were 88.9% versus 52.2% (P=0.01), 100% versus 82.6% (P=0.09), and 100% versus 87.0% (P=0.17), respectively. Conclusions: Increasing sample volume using a 21-gauge needle rather than a 22-gauge needle might improve diagnostic yield in EBUS-TBNA. This study revealed the benefits of using a 21-gauge needle for cytological and histologic diagnostic yields.