Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4+ and CD8+ T lymphocytes and analyze their phenotype as well as the proliferation by flow cytometry in the blood and spleen. We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.