Epstein-Barr virus (EBV) BKRF4 gene product is a tegument protein encoded by a gene with no sequence homology outside of the gamma subfamily of Herpesviridae. Its positional homologs are necessary for an efficient viral lytic program, in particular viral progeny egress and primary infection. To characterize BKRF4 in this regard, EBV recombinant viruses deficient for BKRF4 were developed using site-directed mutagenesis and a bacterial artificial chromosome (BAC)-based recombineering system. Stable human embryonic kidney (HEK) 293 cell lines containing these genomes were generated and the phenotypes of these mutants were analyzed following stimulation of the viral lytic cycle. During the lytic program, BKRF4-null cell lines showed decreased protein expression of various EBV lytic genes that were analyzed using immunostaining and flow cytometry. Reduced amounts of extracellular viral progeny were observed when quantified by real-time PCR and infectivity assays as compared to wild type. These findings suggest an active role of BKRF4 in EBV infection, possibly in viral egress.