EPO normally functions by activating the EPOR, a single-pass transmembrane cytokine receptor required for erythroid differentiation and red blood cell production. TC2-3, the traptamer that activates the hEPOR, supports limited erythroid differentiation in primary human hematopoietic progenitor cells in vitro in the absence of EPO. TC2-3 consists of a 19-amino acid random transmembrane segment flanked by 25 amino acids from E5, forms a homodimer, and displays no sequence or biochemical Talatisamine similarity to EPO. TC2-3 does not activate the PDGFbR or the murine EPOR, and the transmembrane domain of the hEPOR is required for TC2-3 action. We reasoned that isolation of a more active mutant of TC2-3 would facilitate the analysis of small transmembrane activators of the hEPOR and allow the identification of specific features of these proteins that are important for their activity. Here, we used a directed evolution approach to isolate a mutant of TC2-3 with increased activity. A library encoding thousands of TC2-3 mutants was subjected to selection under stringent conditions to isolate a traptamer with enhanced activity, EBC516, which contains a single amino acid substitution that increases dimerization. When expressed in hHPCs, EBC5-16 induces cellsurface expression of the erythroid-specific, differentiation marker, glycophorin A,Crassicauline-A to the same extent as in cells stimulated with EPO. These results suggest that dimerization of EBC5-16 plays a key role in its ability to induce erythroid differentiation. As a first step in understanding the molecular basis for the activity of EBC516, we conducted genetic analysis to identify and characterize its homodimer interface. These experiments provide evidence that increased dimerization of EBC5-16 is responsible for its enhanced activity. This work represents a novel approach to isolate and characterize potent, specific, biologically active proteins not found in nature, which have the potential to modulate the activity of a diverse array of cellular transmembrane proteins of research and clinical importance. In addition, study of these proteins will provide insight into protein-protein interactions occurring in membranes.