Background: The spermatogonial transplantation experiment can be used as an unequivocal detection assay of spermatogenic stem cells (SSCs) in both a qualitative and quantitative manner, based on their regenerative capacity. In this study, the proliferative patterns and kinetics of donor-derived GFRα1-positive spermatogonia containing potential SSCs were examined during early colonization following spermatogonial transplantation.Results: Donor-derived GFRα1-positive cells frequently formed several aggregates of Aal(aligned)/morula-like structures in a single spermatogenic cell patch before and on day 14 post-transplant, indicating a possible involvement in the formation of a stable spermatogenic colony at 21 days post-transplant. The appearance of these Aal/morula-like aggregates is positively correlated with regional, high-level expression of immunoreactive GDNF signals, a ligand for GFRα1, associated with colony expansion. Conclusions: These data raise the hypothesis that regional GDNF signals regulate the balance between donor-derived Aal-like cell aggregates and their differentiation in each small patch, which subsequently leads to further selection of survival colonies at later stages. Developmental Dynamics 241:1374–1384, 2012. © 2012 Wiley Periodicals, Inc.