Single nucleotide polymorphisms (SNPs) in gene encoding pro- and anti-inflammatory factors have been associated with the occurrence of aGvHD. We retrospectively tested a wide panel of 38 polymorphisms in 19 immunoregulatory genes, aiming to first establish, in a pediatric HSCT setting, which SNPs were significantly associated with the development of aGvHD. A significant association was found between aGvHD grades II-IV and SNPs of donor IL10-1082GG, and Fas-670CC + CT and recipient IL18-607 TT + TG genotype. aGvHD grades III-IV resulted associated with donor IL10-1082GG, Fas-670CC + CT, and TLR4-3612TT as well as the use of peripheral CD34+ cells as stem cell source. The multivariate analysis confirmed the association between donor IL10-1082GG and Fas-670CC + CT and aGvHD grades II-IV and between donor IL10-1082GG and TLR4-3612TT and aGvHD grades III-IV. In conclusion we found an association between IL10, FAS, and TLR4 in the donor and IL18 in the recipient and an increased risk of developing aGvHD in transplanted children. Knowledge of the SNPs of cytokine genes associated with aGvHD represents a useful tool for an integrated pretransplantation risk assessment and could guide the physicians to an optimal and more accurate HSCT planning.