Background and Objectives Cell volume changes are hallmarks of both cell maturation and apoptosis, and are paralleled by modulation of membrane ion transport pathways. We evaluated the possible role of K-Cl co-transport (KCC) in both normal and β-thalassemic erythropoiesis in vitro. Design and Methods We studied the effects of the KCC inhibitor, DIOA, on cell proliferation and differentiation, on expression of KCC mRNA and polypeptides, and on expression of cell cycle and apoptosis genes in in vitro liquid-cultures of CD34+ cells from normal and β-thalassemic subjects. Results β-thalassemic erythroid precursors showed increased abundance of KCC1-3 mRNA and of KCC polypeptides in late erythropoiesis. DIOA markedly modified the composition of normal erythroid precursors, promoting differentiation and arrest at the polychromatic erythroblast stage and resulting in a precursor distribution profile similar to that of untreated β-thalassemic cells. DIOA up-regulated cyclin-D mRNA levels in late erythropoiesis in both cell models, paralleling changes in the percentage of S-phase-cells. Caspase-3 activity in late erythropoiesis declined to similar degrees in both cell models. DIOA did not modify caspase-3 or-8 mRNA levels. Interpretation and Conclusions Ineffective erythropoiesis of in vitro cultured β-thalassemic cells is likely related to impaired cell maturation. KCC activity appears to contribute to erythroid cell growth during late erythropoiesis.