Despite transformative advancement in the last decades in cancer immunotherapy, there is clearly a gap in our understanding as not all cancers and not all patients respond to these treatments, which focus mainly on targeting of specific markers expressed by tumor cells, the suppression or enhancement of specific effector cell receptors or the engineering of targeting cytotoxic agents. Treg cells are suppressor T cells which in homeostasis curb autoimmune responses and contribute to the resolution of inflammation, which play a major role in tumor immunity as they were shown to promote tumor growth by the suppression of tumor infiltrating effector cells. Treg relevance to immunotherapy has recently skyrocketed due to the identification of specific intratumoral-Treg markers, which allow a promising safe method of targeting Tregs for cancer therapy while avoiding systemic autoimmune side effects. However, tumor evasion mechanisms interfere with such treatments, as compensatory programs ensue soon after Treg depletion. These compensatory programs are not limited to tumor cells, but also occur in the stromal cells composing the tumor microenvironment (TME), which provide indispensable support to the tumor. Therefore, our goal is to study and characterize the entire spectrum of interactions of Treg cells in the TME, in order to better understand the various levels of regulation and compensation that happen in the context of a developing tumor and stromal cell components, towards designing a new class of Treg TME-focused rational combination immunotherapy treatments.