In the past year we have made important progress towards understanding the role of the Ephs and ephrins classes of signaling molecules in the regulation of hESC growth and differentiation by creating novel tools to detect their expression. These families are large and often multiple members are expressed together during the development of many tissues in the developing mouse. We have determined that many of the 22 members of this large gene family are expressed in human embryonic stem cells hESCs and found that some change their expression upon differentiation of hESCs into human neurons. This leads to the idea that there will be an Eph/ephrin code that can be used to sort and purify specific cell types. We have made several anti-human Eph and ephrin antibodies that recognize the human protein. We have used these antibodies to localize the protein in human stem cells and their differentiated counterparts and determine which types of cells they are expressed in using co-localization with marker proteins. These antibodies will allow us to further probe the function of these proteins. We have perturbed Eph and ephrin function in hESCs using agonists and not detected any difference in cell behavior upon differentiation.