Abstract This study tested the hypothesis that PI-PLCβ1 is associated with myeloid differentiation and that its expression could be useful for predicting the response of MDS patients to azacitidine, as the clinical effect of epigenetic treatments is often detectable only after several cycles of therapy. To this end, PI-PLCβ1 was quantified on 70 MDS patients (IPSS risk: 13 Low, 20 Int-1, 31 Int-2, 6 High) at baseline and during the first 3 cycles of azacitidine. Results were then compared with the hematologic response, as assessed after the sixth cycle of azacitidine therapy. Overall, 60 patients completed 6 cycles of azacitidine, and for them, a clinical and molecular evaluation was possible: 37 of these patients (62%) showed a specific increase of PI-PLCβ1 mRNA within the first 3 cycles, which was associated with a longer duration of response and with an increased myeloid differentiation, as evidenced by PI-PLCγ2 induction and the recruitment of specific myeloid-associated transcription factors to the PI-PLCβ1 promoter during azacitidine response. Moreover, the increase of cyclin D3 gene expression throughout all of the therapy showed that PI-PLCβ1-dependent signaling is indeed activated in azacitidine responder patients. Taken together, our results show that PI-PLCβ1 quantification in MDS predicts the response to azacitidine and is associated with an increased myeloid differentiation.