The recently described JAK2 V617F mutation, present in a substantial proportion of nonchronic myelogenous leukemia chronic myeloproliferative disorders (non-CML CMPDs), is changing the way we conceptualize and diagnose these diseases. We hypothesized that the activation of this tyrosine kinase might result in activation of downstream mediators such as STAT5, which would be detectable in bone marrow biopsies. We examined the expression of activated STAT5 (nuclear phospho-STAT5) in 73 bone marrow biopsies from patients with CMPDs [20 essential thrombocythemia (ET), 26 chronic idiopathic myelofibrosis (CIMF), and 27 polycythemia vera] and 39 controls. We compared the results with the JAK2 mutational status and clinical parameters. The frequency of the JAK2 V617F was 73% (85% in PV, 65% in ET, and 65% in CIMF). All patients with the JAK2 V617F showed abnormal nuclear megakaryocytic phospho-STAT5 (nMEG pSTAT5) expression. In the JAK2 wild-type group, nMEG pSTAT5 was observed in 2/7 ET, and 3/9 CIMF patients. nMEG pSTAT5 staining was 100% sensitive and 88% specific for JAK2 V617F. Clinically, nMEG pSTAT5+ patients seemed to require cytoreductive therapy more often than those without nMEG p-STAT expression. pSTAT5 immunohistochemistry is a useful diagnostic test in bone marrow biopsies from suspected non-CML CMPD patients. It identifies most of the patients with the JAK2 V617F but also other JAK2 wild-type CMPD patients. The presence of nMEG pSTAT5 in a subset of CMPD patients lacking the mutation suggests that alternate tyrosine kinase/phosphatase pathways may be involved and warrant further investigation. Phosphoprotein detection represents a new area for diagnostic pathology that exploits specific functional characteristics of cells within the context of a tissue section.