Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000). We now report that LMP1 drives bfl-1 promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-kappa B dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increased bfl-1 mRNA levels and an NF-kappa B-dependent increase in bfl-1 promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-kappa B-like binding motif that is essential for transactivation of bfl-1 by LMP1, CD40, and the NF-kappa B subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties.