Marek's disease is a highly contagious lymphoproliferative disorder of chickens caused by an oncogenic and strictly cell-associated alphaherpesvirus known as Marek’s disease virus (MDV). MDV is prevalent worldwide and causes fatal lymphomas in the chicken, resulting in a high economic burden. Despite the widespread use of live attenuated vaccines, MDV remains a major pathogen of poultry and continues to be a threat to poultry health and welfare. It has been widely assumed that MDV initially infects B cells, which are the primary target cells in an infected chicken. MDV is then subsequently passed to T cells where it is able to establish a latent infection by integrating its genome into the host telomeres. This integration is a prerequisite for T cell transformation, tumorigenesis and a fatal outcome for the infected chicken. The complex viral processes underlying MDV infections in poultry leading to T cell transformation and lymphomagenesis involve a plethora of viral factors ranging from viral proteins to non-coding RNAs. In order to further our understanding of MDV pathogenesis, I set out to define the exact contribution of specific lymphocytes towards MDV pathogenicity in vivo and uncover the proteomic makeup of MDV-transformed cells. Furthermore, this thesis presents an up-to-date review on the advances in MDV research with a specific focus on its virulence factors. To directly assess the role of B cells in MDV pathogenesis, I utilized the first targeted knockout chickens (JH-KO) that lack mature and peripheral B cells in an in vivo MDV challenge study. These data broke the dogma regarding the vital role of B cells in MDV pathogenesis, and demonstrated that they are completely dispensable for virus replication, spread in the host, disease and tumor development. Moreover, it was shown that CD4+ and CD8+ T cells complement for the loss of B cells in JH-KO chickens in terms of virus amplification and virus spread in the host. Secondly, advances in tumor imaging and mass spectrometry allowed acquisition of MDV-tumor proteomic data. This thesis describes the establishment and implementation of an imaging mass spectrometry (IMS)-based pipeline that was used to identify potential protein biomarkers of MDV-induced lymphomas. IMS and subsequent non-contact laser capture microdissection of MDV lymphoma was followed by a proteomic workflow and provides an unbiased ‘open view’ tool for protein mass spectrometry of MDV-induced tumors. Lastly, this thesis provides a review of all recent literature and advances in MDV research on virus virulence factors. This summarizes the current scientific consensus of how viral factors contribute to MDV-induced pathogenesis and tumor formation. Several important viral factors involved in MDV pathogenesis have been discussed, including the major oncoprotein Meq, the viral chemokine vIL-8, MDV-encoded microRNAs, RLORF4, RLORF5a, pp14, pp38, a virus-encoded telomerase RNA, and viral telomeric repeats. Overall, this thesis contributes towards a greater understanding of MDV pathogenesis, shedding light on the cell types involved in virus replication and spread in vivo and factors present in MDV-induced tumors.