<jats:title>Abstract</jats:title><jats:p>The clinical success of immune checkpoint modulators and the development of next‐generation immune‐oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (<jats:italic>Prkdc<jats:sup>scid</jats:sup> Il2rg<jats:sup>tm1Sug</jats:sup></jats:italic>) and triple‐transgenic NOG‐EXL [<jats:italic>Prkdc<jats:sup>scid</jats:sup> Il2rg<jats:sup>tm1Sug</jats:sup></jats:italic> Tg (SV40/HTLV‐IL3, CSF2)], which expresses human IL‐3 and GM‐CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post‐transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T‐cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC‐HIS model can be limited due to onset of xenogeneic graft‐versus‐host disease (xGVHD) within 3 to 5 weeks post‐implantation. Host deficiency in MHC class I, as occurs in beta‐2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood–derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC‐HIS and PBMC‐HIS mouse models are provided. In addition, the co‐engraftment and growth kinetics of patient‐derived and cell line–derived xenograft tumors in humanized mice and recovery of tumor‐infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors.</jats:p><jats:p><jats:bold>Basic Protocol 1</jats:bold>: Establishment of patient‐derived xenograft tumors in CD34+ hematopoietic stem cell–humanized mice</jats:p><jats:p><jats:bold>Basic Protocol 2</jats:bold>: Establishment of patient‐derived xenograft tumors in peripheral blood mononuclear cell–humanized mice</jats:p><jats:p><jats:bold>Support Protocol 1</jats:bold>: Flow cytometry assessment of humanization in mice</jats:p><jats:p><jats:bold>Support Protocol 2</jats:bold>: Flow cytometry assessment of tumor‐infiltrating lymphocytes in tumor‐bearing humanized mouse models</jats:p>