In the bi-directional signaling system comprising ephrins (EFNs) and ephrin receptors (Ephs), both EFNs and Ephs simultaneously function both as ligands and as receptors. Importantly, the EFN/Eph system is deregulated in human cancers and has been implicated in the metastatic processes because of its effects on the adhesion and migration of epithelial cells. The idiosyncratic function of Ephs, membrane-bound receptor kinases, as extracellular signaling ligands, has not been extensively studied. This prompted us to explore the transcriptional targets regulated by Ephs acting solely as ligands. To define the ligand function of EphB2 in human epidermal keratinocytes, we treated these cells with EphB2 as Fc-conjugate dimmers, which thus act exclusively as extracellular ligands. We compared the EphB2 and EFNA4 effects during a 48 h time course, using transcriptional profiling. We found that EphB2, acting as a ligand, promotes epidermal differentiation. For example, EphB2 induces expression of markers of epidermal differentiation, including keratins KRT1 and KRT10, SPRRs, desmosomal proteins and cell cycle inhibitors, while suppressing basal layer markers, integrins and cell cycle proteins. The effects of EphB2 are delayed relative to those of EFNA4. Unlike EFNA4, EphB2 did not induce lipid metabolism proteins, this particular aspect of epidermal differentiation seems not to be regulated by EphB2. Our results define the transcriptional targets of the reverse signaling by EphB2 acting exclusively as a ligand and begin to characterize this intriguing function of Ephs.