Constitutive tyrosine kinase activity of the breakpoint cluster region (Bcr)-Abl fusion protein is characteristic of chronic myelogenous leukemia (CML). As resistance against Imatinib a Bcr-abl inhibitor used in CML, was described, Heat shock protein (Hsp90) became an alternative target as inhibition of Bcr-Abl-Hsp90 complex leads to proliferation arrest. Here, we used natural product Radicicol (Rad), a macrocyclic antifungal, as an Hsp90 inhibitor to investigate the effect of Bcr-Ab1 inactivation on erythroid gene expression and subsequently on the transcription factors involved in their regulation. We showed that all erythroid genes studied were over-expressed after Rad treatment while Bcr-Abl expression was inhibited. Specific transcription factor NF-E2 was induced in Rad-treated cells as well as GATA-1 cofactors Friend of GATA (FOG)1 and SP1, whereas PU.1 was downregulated. Moreover, p38 mitogen activated protein kinase (MAPK) inhibition prevented Rad-mediated differentiation of K562 in correlation with decreased γ-globin expression and suppression of Rad-mediated inhibition of PU.1. In conclusion, our results show that Radicicol leads to Bcr-Abl inactivation via Hsp90 inhibition inducing reactivation of the erythroid program in K562 cells.