The TR2 and TR4 orphan nuclear receptors comprise the DNA-binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal beta-type globin gene promoters. Silencing of both the embryonic and fetal beta-type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant-negative TR4 (dnTR4), human embryonic epsilon-globin is activated in primitive and definitive erythroid cells. In contrast, human fetal gamma-globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage-selective repressor. Forced expression of wild-type TR2 and TR4 leads to precocious repression of epsilon-globin, but in contrast to induction of gamma-globin in definitive erythroid cells. These temporally specific, gene-selective alterations in epsilon- and gamma-globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene-autonomous silencing of the epsilon- and gamma-globin genes during development, and suggest that their differential utilization controls stage-specific repression of the human epsilon- and gamma-globin genes.