Targeted immunotherapies have revolutionized outcomes for lymphoid malignancies, but success in myeloid neoplasms is limited by the lack of amenable targets and immunologically hostile tumor microenvironment (TME). Myeloproliferative neoplasms (MPNs) are chronic myeloid blood cancers, a third of which are driven by mutations in calreticulin. Calreticulin mutant protein (mutCALR) binds and activates thrombopoietin receptor (TpoR) and results in the display of the mutCALR-TpoR complex on the extracellular membrane of disease-driving cells, thus exposing a therapeutic vulnerability. Here, we present a chimeric antigen receptor T cell (CAR T cell) therapy that specifically targets mutCALR+ cells, both in vitro and in vivo. The CAR T cell therapy selectively depleted mutCALR+ stem cells in samples from patients with myelofibrosis without affecting healthy stem cells and improved survival in mutCALR leukemia xenograft models. To mimic myelofibrotic marrow, we developed a human “chimeroid” model and showed no decrease in the potency of CAR T cell–mediated target cell killing even in a fibrotic TME. We also devised a method to boost the cell surface expression of mutCALR in CD34+ cells isolated from patients with accelerated/blast phase MPNs (defined as >10% blasts in the peripheral blood or bone marrow), enhancing CAR T cell targeting. This study presents a therapeutic strategy with potential to eradicate mutCALR-driven malignancies and highlights an innovative strategy to evaluate blood cancer–targeting immunotherapies in a relevant TME.