Regulatory T cells (Tregs) suppress other immune cells and, thus, are critical mediators of peripheral self-tolerance, preventing autoimmune disease but hampering tumor rejection. Therapeutic manipulations of Treg number and function are therefore subject to numerous clinical investigations. First in-man trials of adoptive Treg transfer to prevent graft-versus-host disease showed very promising outcomes. Yet, the number of naturally occurring Tregs (nTregs) is minute, encouraging the complementary approach of inducing Tregs (iTregs) from naive T cell precursors. Reinforcing this concept, there are several studies in mice indicating that iTreg transfer may be superior to nTreg transfer. Moreover, iTregs are generated in vivo and ample evidence corroborates that iTregs exert non-redundant functions to maintain health. However, the molecular mechanisms governing iTreg generation in humans are incompletely understood and procedures for human iTreg generation are controversial. Here we will therefore establish and compare different protocols of human iTreg generation juxtaposed with deep molecular profiling using RNA sequencing and DNA methylation analysis. The time-dependent transcriptomic and epigenetic data, capturing molecular events during generation of iTregs, will be subject to bioinformatics analysis. Our integrative analysis will present the first global hierarchical molecular map of pathways driving human iTreg differentiation, which in addition will provide a molecular scaffold enabling deeper analyses to resolve molecular events independent and dependent of established factors. Furthermore, the biological role of novel transcripts, splice variants and epigenetic regulations will be validated biologically by gene knockdown or overexpression experiments. Our interdisciplinary analysis of the molecular mechanisms ruling human iTreg generation may have important implications for our understanding and ability to treat cancer, autoimmune and inflammatory diseases.