Humanized mouse models of graft-versus-host disease (GVHD), involving the injection of human immune cells into immunodeficient mice, are firmly established and offer avenues for exploring pathways implicated in GVHD development and for testing therapeutics to prevent or treat this disease. NOD-scid-IL-2Rγnull (NSG) mice are a strain of immunodeficient mice commonly used to study GVHD. This chapter details methods relating to this model including human immune cell isolation, the injection of these human immune cells into nonirradiated NSG mice, monitoring the mice for signs of clinical GVHD (including weight loss), immunolabeling with anti-human and anti-mouse monoclonal antibodies of leukocytes isolated from the spleens, livers, and lungs of humanized mice, evaluating human cell engraftment and human and mouse immune cell subsets via flow cytometry and measuring serum cytokine concentrations using a multiplexed flow cytometric assay.