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Abstract Introduction Invariant natural killer T cells (iNKTs) are a rare but vital subset of immunomodulatory T cells and play an important role in allogeneic hematopoietic stem cell trans-plantation (HSCT). The association of donor characteristics with the number and frequency of the iNKTs subsets in allografts remains poorly understood. In this paper, we prospectively investigate the association of donor characteristics with iNKTs dose and frequency in granulocyte–colony-stimulating factor (G-CSF) mobilized marrow and peripheral blood harvests. Materials and methods 100 bone marrow (BM) units and 100 peripheral blood (PB) units from 100 healthy donors were examined. Parameters including donor age, sex, weight, height, BMI and blood count [including white blood cells (WBCs), lymphocytes and monocytes] at three time points [donor's steady state before G-CSF administration, the day of G-BM harvesting and the day of G-PB apheresis] were analyzed to explore the impact of donor characteristics on iNKTs composition in BM and PB grafts. Results Multivariate analysis showed monocyte counts before G-BM harvest could predict higher frequency of iNKTs in WBC (OR = 2.593, 95%CI: 1.128–5.961, p = 0.025), higher total CD4+ iNKTs dose (OR = 2.250, 95%CI: 1.011–5.008, p = 0.047) and higher total iNKTs dose (OR = 2.662, 95%CI: 1.187–5.970, p = 0.017) in mixture allografts. Discussion The results suggested that monocyte counts pre G-BM harvest could predict the yield of total CD4+ iNKTs and total iNKTs in mixture allografts. The male and older donors were associated with a higher dose of total CD4− iNKTs in mixture allografts. Highlights • Monocyte before G-BM harvest predicted iNKT/WBC, iNKT/T and iNKTs dose in allograft. • Weight and monocyte pre-mobilization were positively associated with iNKT/T. • Allografts from male and elder donors contained higher dose of CD4− iNKTs. • 100 G-BM and 100 G-PB units from 100 healthy donors examined in this study. [ABSTRACT FROM AUTHOR]