The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 173% (17/98) of normal donors; the mutant allele burden was, however, very low (1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34(+) cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F. (C) 2017 Elsevier Inc. All rights reserved.