Simple Summary Immunotherapies have revolutionized the field of oncology and have been approved to treat cancer. Despite progress in immunotherapy, many challenges remain, including the identification (i) of predictive markers for treatment response or (ii) of beneficial T cell subsets involved in tumor elimination. "Humanized" mice are a promising translational model for studying the human immune system in the context of immuno-oncology research. Here, multicolor flow cytometry was applied to characterize immune cell subsets in the spleen of humanized mice transplanted with patient-derived breast cancer tissues. This multicolor immune cell setup will help to identify promising therapeutic approaches or predictive immune cell subsets in the future using humanized tumor mice. "Humanized" mice have been widely used for the characterization of human cancer progression and as a powerful preclinical model. Standardization of multicolor phenotyping could help to identify immune cell patterns involved in checkpoint-related complications. Therefore, we applied established protocols for immune cell profiling to our humanized Patient-Derived Xenograft (hPDX) model. hPDX are characterized by the co-existence of a human immune system and a patient-derived tumor transplant. These mice possess a human-like immune system after CD34(+) stem cell transplantation while the reconstitution level of the immune system was not related to the quantity of transplanted CD34(+) cells. Contamination <= 1.2% by CD3(+) cells in the hematopoietic stem cell (HSC) transplant did not trigger abnormal T cell maturation. Different B and T cell differentiation stages were identified, as well as regulatory T cells (Tregs) and exhausted T cells that expressed TIGIT, PD-1, or KLRG1. Overall, the application of standardized protocols for the characterization of immune cells using flow cytometry will contribute to a better understanding of immune-oncologic processes.