Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown thathbzmRNA promotes T-cell proliferation. In our current studies, we evaluated the role ofhbzmRNA on HTLV-1-mediated immortalizationin vitroas well asin vivopersistence and disease development. We generated mutant proviral clones to examine the individual contributions ofhbzmRNA,hbzmRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cellsin vitro. Viral persistence and disease development were also evaluatedin vivoby infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an alteredhbzmRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. AlteredhbzmRNA secondary structure, or loss ofhbzmRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesisin vivo.