Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for a variety of blood disorders but primarily reserved for blood cancer patients due to the life-threatening risk of acute graft-versus-host disease (aGVHD). Development of aGVHD is driven by alloreactive T cells that recognize the recipient’s tissues as foreign and initiate a robust immune response causing severe tissue damage of the skinSkin, liver, and gut. While methods to reduce aGVHD incidence and severity exist including posttransplant cyclophosphamide, relapse rates and transplantation-related mortality remain. We previously published a study using graft conditioning with glucocorticoids as a strategy to modify the immune repertoire in the donor grafts prior to transplantation to reduce aGVHD. Here, we describe a protocol to characterize the T-cell response in recipient mice given transplantation of graft-conditioned allogeneic donor cells via mass cytometry. Mass cytometry is a technology that uses heavy metal tags in place of fluorochromes to allow high-dimensional flow cytometric analysis. In this protocol, we include an antibody panel of 39 different antibodies conjugated to 41 different heavy metal tags that have been validated and titrated for barcoding and immunophenotyping by lineage markers, activation markers, cytokine secretion, and transcription factors. This chapter details the graft conditioning, transplantation, processing, barcoding, staining, and mass cytometric analysis of immune cells following murine allo-HCT.