Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185–211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1Δ185–211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1Δ185–211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.