JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/β-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/β-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/β-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/β-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/β-catenin signalling as a potential therapeutic target for MPN.© 2023 British Society for Haematology and John Wiley & Sons Ltd.