Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4⁺CD25^highCD127⁻ Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and in vitro expansion capacity after SC mobilization. Most importantly, in vivo G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after in vitro expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.