Abstract Objective Allogeneic transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) -mobilized peripheral blood stem cells (PBSCs) is now being increasingly performed, but safety considerations for hematologically normal PBSC donors have not been fully addressed. The effection of G-CSF on donors’ lymphocytes have not been defined clearly. Our study was to detect and analyze the phenotypical and functional properties of lymphocytes from allo-PBSC donors treated with recombinant human G-CSF by flow cytometry. Methods Thirty-four HLA-identical sibling donors (13male, 21female; median age, 35 years) were treated by subcutaneous injection with rhG-CSF at a dose of 5 μg/kg twice daily for 4–6 consecutive days to mobilize HSCs to the peripheral blood. Leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra, Lakewood, CO) on 1 to 2 consecutive days beginning on day 4 of rhG-CSF administration. Donor blood samples, which were obtained before the first administration of G-CSF (pre-G), on day 4 of G-CSF administration (post-G), and one week after the last rhG-CSF were analyzed by 3-color flow cytometry. Monoclonal antibodies included: CD3, CD4, CD8, CD20, CD16, CD56, CD25, CD69, CD45RA, CD45RO, CD28, CD95. Results Absolute counts of lymphocytes, B cells, T cells, CD4+ and CD8+cells post-G were significantly elevated two more times than pre-G (P<0.05), but these changes recovered when the last G-CSF administrated one week later. The percentage of CD3+, CD4+, CD8+ and the ratio of CD4 and CD8 T cells had no significantly difference between pre-G and post-G. The percentage of CD4+CD25+, CD4+CD45RO+ and CD4+CD28+ T cells were significantly decreased post-G (45.26±9.68% to 37.34±11.12%; 65.53±13.74% to 52.32±13.47%; 94.75±4.02% to 91.74±8.72%, P=0.00329, 0.0003 and 0.0947, respectively). We found that CD4+CD28+ T cells(91.77 ± 6.15, P=0.00218) were still lower than pre-G when stopped administrate G-CSF one week later, while others changes have recovered to the Pre-G level. Conclusion: The study shows that CD4+CD28+ T lymphoctye subsets were still lower than pre-G, although most of other change recover one week later after mobilization