In addition to IgE receptor (FcεRI), a subtype of human mast cells (MCs) express Mas-related G protein-coupled receptor-X2 (MRGPRX2, mouse ortholog MrgprB2). Studies using MrgprB2−/− mice have shown that this receptor is involved in anaphylaxis, asthma, neurogenic inflammation, and a variety of cutaneous disorders. Although MrgprB2 and MRGPRX2 display similar tissue distribution, they have ~53% sequence identity with distinct pharmacologic properties. We hypothesized that newly identified small-molecule MRGPRX2 antagonists (C9 and C9-6) can selectively inhibit MRGPRX2 without affecting FcεRI or MrgprB2-mediated responses, and that establishing preclinical mouse models expressing MRGPRX2 in MCs would enable us to study MRGPRX2 modulation in vivo. For this, our first goal was to determine the specificity of C9 and C9-6 for MC degranulation. We found that C9 and C9-6 selectively inhibited MRGPRX2 but not antigen/IgE or MrgprB2-mediated MC degranulation. We utilized two approaches to replace endogenous mouse MrgprB2 with human MRGPRX2 for in vivo studies. First, we used a retrovirus to express MRGPRX2 in bone marrow (BM) cells obtained from MrgprB2−/− mice. These cells were differentiated ex vivo into BMMCs, and their functional properties were confirmed by testing the ability of specific agonists to induce Ca2+ mobilization, degranulation, and TNF-α generation before they were engrafted intraperitoneally into MC-deficient Wsh/Wsh mice. MCs recovered from peritoneal lavage of engraftment mice retained cell surface expression of MRGPRX2 and responded to substance P (SP) for degranulation. For the second approach, we utilized the CRISPR/Cas9 gene editing approach to generate MRGPRX2 knock-in (KI) mice. Using double immunofluorescence staining, we found that MRGPRX2-expressing MCs are present in the skin, gingiva, trachea, and colon of MRGPRX2-KI mice, like the situation in human tissues. Furthermore, most agonists tested induced greater degranulation at lower concentrations in peritoneal MCs from MRGPRX2-KI mice compared to MrgprB2-expressing cells. Finally, we sought to determine if C9 and C9-6 would modulate IgE-mediated systemic anaphylaxis in vivo. While both inhibited degranulation in response to SP, it had no effect on IgE-mediated degranulation in PMCs in vitro but attenuated systemic anaphylaxis in MRGPRX2-KI mice. These data demonstrate the novel finding that MRGPRX2 contributes to systemic anaphylaxis through its transactivation, likely via mediators released from IgE-mediated MC activation. Thus, the establishment of preclinical models that express MRGPRX2 in MCs in mice provides an essential tool for exploring MRGPRX2 signaling and opens avenues for testing novel therapeutics for MC-mediated disorders.