Metazoan development is regulated by transcriptional networks, which must respond to extracellular cues including cytokines. The JAK/STAT pathway is a highly conserved regulatory module, activated by many cytokines, in which tyrosine‐phosphorylated STATs (pSTATs) function as transcription factors. However, the mechanisms by which STAT activation modulates lineage‐affiliated transcriptional programs are unclear. We demonstrate that in the absence of thrombopoietin (TPO), tyrosine‐unphosphorylated STAT5 (uSTAT5) is present in the nucleus where it colocalizes with CTCF and represses a megakaryocytic transcriptional program. TPO‐mediated phosphorylation of STAT5 triggers its genome‐wide relocation to STAT consensus sites with two distinct transcriptional consequences, loss of a uSTAT5 program that restrains megakaryocytic differentiation and activation of a canonical pSTAT5‐driven program which includes regulators of apoptosis and proliferation. Transcriptional repression by uSTAT5 reflects restricted access of the megakaryocytic transcription factor ERG to target genes. These results identify a previously unrecognized mechanism of cytokine‐mediated differentiation.