Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets are particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signaling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions. This article is protected by copyright. All rights reserved.