Vaccination is an effective strategy to reduce the loss of chickens in the poultry industry caused by Marek's Disease (MD), an avian lymphoproliferative disease. The vaccines currently used are from attenuated serotype 1 Marek's disease virus (MDV) or naturally nononcogenic MDV strains. To prepare for future immunity breaks, functional genomic and proteomic studies have been used to better understand the underlying mechanisms of MDV pathogenicity and the effects induced by the vaccine viruses. In this study, a combined approach of quantitative GeLC-MSE and qualitative ERLIC/IMAC/LC-MS/MS analysis were used to identify abundance changes of proteins and the variations of phosphorylation status resulting from the perturbations due to infection with an attenuated oncogenic virus strain (Md11/75C) and several nononcogenic virus strains (CVI988, FC126 and 301B) in vitro. Using this combined approach, several signal transduction pathways mapped by the identified proteins were found to be altered at both the level of protein abundance and phosphorylation. On the basis of this study, a kinase-dependent pathway to regulate phosphorylation of 4E-BP1 to modulate assembly of the protein translation initiation complex was revealed. The differences of 4E-BP1 phosphorylation patterns as well as the measured abundance changes among several other proteins that regulate host transcriptional and translational activities across the virus strains used in this study provide new insight for future functional and biochemical characterization of specific proteins involved in MDV pathogenesis.