Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2'-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.