Up-regulation of tumor necrosis factor alpha (TNFalpha) is linked to solid tumors as well as to hematologic disorders including different forms of anemia and multiple myeloma. This cytokine was shown to contribute to inhibition of erythroid maturation mechanisms which are characterized by the expression of specific genes regulated by GATA-1 and NF-E2 transcription factors. Here, we assessed the inhibiting effect of TNFalpha on erythroid differentiation using K562 cells which can be chemically induced to differentiate towards the erythroid pathway by aclacinomycin A, an anthracyclin. Results show that induced hemoglobinization of K562 cells as well as gamma-globin and erythropoietin receptor gene expression are decreased by TNFalpha via the inhibition of GATA-1 at its mRNA and protein expression level. Additionally, both constitutive and induced binding activity of GATA-1 is abolished and induced activation of a GATA-1 driven luciferase reporter construct is inhibited. Altogether, our results provide insight into the molecular mechanisms of inflammation-induced inhibition of erythroid differentiation.