Globin gene switching is a well-described model of eucaryotic developmental control. In the case of the human alpha-globin gene cluster, migration of erythropoietic activity from the embryonic yolk sac to the fetal liver is parallaled by the zeta-globin gene silencing and enhanced expression of the alpha-globin genes. To map critical cis determinants of this switch, the human zeta-globin gene, the alpha-globin gene, and chimeric recombinants were introduced into the mouse genome. Consistent with previous studies, expression of the individual alpha- and zeta-globin transgenes was found to be developmentally appropriate. Contrary to current models, however, the alpha- and zeta-globin gene promoters were not sufficient to establish this control. Instead, full silencing of the zeta-globin gene required the combined activities of this promoter, transcribed region, and 3'-flanking sequences. Individually, the silencing activities of the zeta-globin gene promoter and 3'-flanking region were minimal but increased markedly when both regions were present. The zeta-globin transcribed region appeared to contribute to gene silencing by a mechanism specifically activated in definitive erythroblasts in the fetal liver. These data demonstrate that a complex set of controls, requiring at least three determinants and involving at least two independent mechanisms, is necessary for full developmental silencing of the human zeta-globin gene.