Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCV) from non-human primates infect B cells, transform their growth to facilitate life-long viral persistence in the host, and contribute to B cell oncogenesis. Co-evolution of LCV with their primate hosts has led to species-specificity so that LCVs preferentially immortalize B cells from their natural host in vitro. We investigated whether the master regulator of transcription, EBV nuclear antigen 2 (EBNA2), is involved in LCV species-specificity. Using recombinant EBVs, we show that EBNA2 orthologues of LCV isolated from chimpanzees, baboons, cynomolgus or rhesus macaques cannot replace EBV EBNA2 for the immortalization of human B cells. Thus, LCV species-specificity is functionally linked to viral proteins expressed during latent, growth-transforming infection. In addition, we identified three independent domains within EBNA2 that act through species-specific mechanisms. Importantly, the EBNA2 orthologues and species-specific EBNA2 domains separate unique roles for EBNA2 in the initiation of B cell immortalization from those responsible for maintaining the immortalized state. Investigating LCV species-specificity provides a novel approach to identify critical steps underlying EBV-induced B cell growth transformation, persistent infection, and oncogenesis.