High affinity, neutralizing antibodies against coagulation factor VIII (FVIII), so called FVIII inhibitors, are the major treatment complication in the therapy of congenital hemophilia A and causative for the rare autoimmune disorder acquired hemophilia A. It is generally accepted, that for the induction of high-affinity antibody responses against protein antigens B cells need the help of activated CD4+ T cells to differentiate into antibody-secreting plasma cells. Both clinical and experimental data support the central role of CD4+ T cells in the regulation of FVIII inhibitors in congenital hemophilia A. However, very little information is available on the evolution of FVIII-specific immune responses in patients during the early phases of FVIII substitution therapy. Additionally, the relevance of FVIII-binding antibodies in healthy individuals and triggers causing the generation of FVIII inhibitors in acquired hemophilia A patients are far from being understood. Our approach to uncover the pluralism of causes and effects in FVIII-specific immune responses is to capture the major players of the adaptive immune system involved, namely antibodies and CD4+ T cells. To be able to study early phases of FVIII-specific immune responses in infant hemophilic patients, the applied technologies have to be optimized for small blood volumes and should be applicable to a multi- center setting of clinical studies. In the course of this PhD project a set of assays was established and validated to extensively characterize antibodies against FVIII and to identify FVIII-specific CD4+ T cell signatures. Antibody assays not only include the differentiation of Ig isotypes and IgG subclasses, but also allow for the assessment of apparent affinities of FVIII-specific antibodies to enable monitoring of affinity maturation of antibodies during the evolvement of FVIII inhibitors. The analysis of FVIII-specific CD4+ T cell signatures, which potentially regulate FVIII inhibitor development, is based on global gene expression via microarray technology after short in vitro restimulation of PBMCs. Proof of principle data published in a peer- reviewed journal and presented at several international scientific conferences exemplifies the analytic power of this approach. The application of our FVIII-specific immune monitoring in prospective, longitudinal studies will help to gain further understanding of the interplay between genetic and environmental influences causing the evolution of anti-FVIII immune responses and the role of antigen specific CD4+ T cells in this system. This valuable information will be integral in efforts to improve hemophilia A care and quality of life for patients afflicted from FVIII inhibitors.