While Human Leukocyte Antigen (HLA) typing remains the central means of selecting stem cell donors and determining stem cell transplantation (SCT) outcome, non-HLA molecules are also involved in the process. HLA compatibility is the main factor determining the occurrence of graft-versus-host disease (GvHD) in patients. It has also been shown that minor histocompatibility antigen differences as well as genetic polymorphisms that are not sequenced by standard methodology for HLA typing can play a role. As early predictive markers for the occurrence of GvHD are still lacking and a better understanding of the mechanisms and pathways involved are still needed to provide insight into the pathogenesis of the disease, this study aims to identify novel targets for identifying allogeneic responses predictive of such a serious complication of this therapeutic treatment. The mixed lymphocyte culture (MLC) model was used under different conditions in order to identify potential genetic markers associated with allogeneic responses. Initially, this model using the mononuclear cell fraction of blood samples from mismatched individuals was validated by comparing cell activation and proliferation and time course studies, as well as using irradiation of cells and lipopolysaccharide (LPS) in an effort to mimic the in vivo conditions occurring during the infusion of allogeneic cells to patients during SCT. Through a limited gene expression analysis of immune related genes, of both HLA matched and HLA-mismatched mononuclear cell fractions, we further validated the time and conditions used for global gene expression profiling and observed different gene expression changes between the two HLA settings. The investigation of global gene expression changes, of the HLA matched and HLA mismatched MLC, enabled us to identify differentially regulated genes and pathways. A great number of these genes were related to immune function. These genes were also found to be associated with GvHD and graft rejection in previous studies. By investigating global gene expression changes in mismatched MLC we aimed to identify genes that may constitute good targets to study further as markers for donorrecipient alloresponses. The most highly upregulated genes were IDO1, CXCL9, CXCL10 and CCL8. The majority of differentially regulated genes were IFN-γ inducible genes and IFN-γ neutralisation in MLCs abrogated their induction. The microRNA-155, a recently identified target for GvHD, was also found to be significantly induced in HLA mismatched MLC but not in the matched setting and its induction was not diminished by IFN-γ blockade. In this proof-of-principle study we show gene expression changes in mismatched MLC that represent alloreactive responses and correlate with markers involved in GvHD and can potentially be useful in further studies of the biological processes involved in this disease but also in the search of early predictive markers for the disease.