An activating point mutation in Janus kinase 2 (JAK2 V617F) was recently identified in myelofibrosis with myeloid metaplasia (MMM). To further elucidate the pathogenic significance, we examined the JAK2 mutation burden, phosphorylation of JAK2 substrates and neutrophil apoptotic resistance. Immunoblotting revealed phosphorylation of signal transducer and activator of transcription-3 (STAT3) in all four JAK2 with high V617F mutant allele burden and seven of eight with intermediate mutant allele burden, but only one of eight with wild-type JAK2 (P<0.001). In contrast, STAT5 phosphorylation was undetectable in patient MMM neutrophils; and phosphorylation of Akt and extracellular signal-regulated kinases (ERKs) failed to correlate with JAK2 mutation status. Apoptosis was lower in MMM neutrophils (median 41% apoptotic cells, n=50) compared to controls (median 66%, n=9) or other myeloproliferative disorder patients (median 53%, n=11; P=0.002). Apoptotic resistance in MMM correlated with anemia (P=0.01) and the JAK2-V617F (P=0.01). Indeed, apoptotic resistance was greatest in MMM neutrophils with high mutant allele burden (median 22% apoptosis, n=5) than with intermediate burden (median 39%, n=23) or wild-type JAK2 (median 47%, n=22; P=0.008). These results suggest that mutant JAK2 contributes to MMM pathogenesis by constitutively phosphorylating STAT3 and diminishing myeloid cell apoptosis.