The formation of functional skin entails multiple key signals that are implicated repeatedly in distinct processes during embryogenesis. Although Eph receptors and their membrane-bound ephrin ligands play a role in a wide variety of embryonic processes, their function in skin development has not been addressed. Here, we show that ephrin B2 is transiently expressed in hair buds during embryogenesis and in dermal mesenchymal cells during the perinatal period. Keratinocyte-specific ephrin B2-targeted mutant mice exhibit no skin phenotype, whereas postnatal systemic ephrin B2 ablation results in the enhancement of keratinocyte proliferation. Although the same treatment results in a defect of vascular remodeling, our analyses showed that the keratinocyte phenotype is not caused by hypoxia due to vascular defects. Interestingly, we found an enhanced expression of IL-1 family molecules, which have been implicated in the regulation of keratinocyte proliferation. On the basis of these observations, we propose that the transient expression of ephrin B2 in perinatal dermal mesenchymal cells plays a role in adjusting the activity of the mesenchymal microenvironment that regulates proliferation of keratinocytes.