Human Leucocyte Antigen (HLA) alleles, cytokine polymorphisms and the type of factor VIII (FVIII) gene mutation are among predisposing factors for inhibitors (inh) development in children with severe haemophilia A (HA). The aim was to investigate the correlations among (i) FVIII gene intron-22 inversion, (ii) HLA alleles and haplotypes and (iii) certain cytokine polymorphisms, with the risk for FVIII inhibitors development in 52 Greek severe HA children, exclusively treated with recombinant concentrates. We performed Long-Range PCR for detection of intron-22 inversion and PCR-SSP, PCR-SSO for genotyping of HLA-A, B, C, DRB1, DQB1 alleles and also for cytokine polymorphisms of TNF-α, TGF-β1, IL-10, IL-6 and IFN-γ. Chi-squared test and Fischer's exact test were used for statistical analysis. A total of 28 children had developed inhibitors (Group I), 71.4% high responding, while 24 had not (Group II). No statistically increased intron-22 inversion prevalence was found in Group I compared with Group II (P = 0.5). Comparison of HLA allele frequencies between the two groups showed statistically significant differences in the following genotypes (i) promoting inhibitors development: DRB1*01(P = 0.014), DRB1*01:01(P = 0.011) and DQB1*05:01 (P = 0.005) and (ii) possibly protecting from inhibitors development: DRB1*11 (P = 0.011), DRB1*11:01 (P = 0.031), DQB1*03 (P = 0.004) and DQB1*03:01 (P = 0.014). Analysis of cytokines revealed a higher incidence of inhibitor detection only in homozygotes of the haplotypes ACC and ATA for IL-10 polymorphisms (P = 0.05). There is evidence that HLA alleles and cytokine polymorphisms play an important role in FVIII inh development. On the contrary, no statistically significant results were obtained for intron-22 inversion and its impact on FVIII inhibitors formation.