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Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. Current nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, limiting the application of this life-saving treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches, such as antibody-drug conjugates, naked antibodies, and CAR T cells. Here, we introduce a preconditioning strategy targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, genomic disruption of the CD45 gene was performed on human CD45 CAR T cells in combination with the signaling kinase inhibitor dasatinib. CD45Δ CAR45 T cells showed high cytotoxicity in vitro and depletion of tumor cells in vivo. These cells were effective in elimination of human hematopoietic cells engrafted in humanized immunodeficient mice by transfusion with human blood-derived hematopoietic stem cells (HSCs). Similarly, CD45Δ CAR45 natural killer (NK) cells exhibited potent cytotoxicity toward tumor cell lines and human hematopoietic cells in vitro. Thus, we provide the proof of concept for the generation and preclinical efficacy of fratricide-resistant CAR45 T and NK cells directed against CD45-expressing tumors and hematopoietic cells.