Differentiation of multipotential hematopoietic cells into lineage-committed precursors involves the selection and maintenance of appropriate programs of gene expression, regulated by specific transcription factors. Using human K562 erythroleukemia cells capable of differentiating along erythroid and megakaryocytic lineages, we explore the differentiation-related role of heat shock transcription factor 2 (HSF2), which belongs to a family of transcription factors generally known to regulate heat shock gene expression. We demonstrate that enhanced HSF2 expression and the acquisition of HSF2 DNA binding activity are strictly specific for erythroid characteristics of K562 cells. Our results reveal a multistep regulatory process of HSF2 gene expression. In K562 cells undergoing hemin-mediated erythroid differentiation, the increase in HSF2 protein levels is preceded by transcriptional induction of the HSF2 gene, accompanied by increased HSF2 mRNA stability. In contrast, during megakaryocytic differentiation induced by the phorbol ester TPA, expression of HSF2 is rapidly down-regulated, leading to a complete loss of the HSF2 protein. These results indicate that the determination of HSF2 expression occurs at the early stages of lineage commitment. Taken together, our data suggest that HSF2 could function as a lineage-restricted transcription factor during differentiation of K562 cells along either the erythroid or the megakaryocytic pathway.