It remains a wonder how Tregs induce tolerance for the development of cancer. Previously we have shown with melanoma patients that, increase in peripherally induced Tregs (pTregs) number in blood is related to the poor prognosis of the disease. In vitro induced Tregs (iTregs) and pTregs are remarkably similar and significantly different in functionality from tTregs. Here, we worked with 12 melanoma patients-six HLA A2 positive and six HLA A2 negative. PBL and tumor cells were obtained from the patients with informed consent. Treg cells were generated and isolated from four different culture conditions: 1) Isolated from tumor +PBL IVC, 2) Mart-1 A2 or 3) Flu A2 pulsed DC + PBL IVC and 4) purified CD4+CD25-cells stimulated with anti CD3 and antiCD28 plus IL-2. We used these different Treg generation conditions (self vs. non-self) to understand how induced Tregs behave phenotypically and functionally that would open number of avenues to over come their negative effects. Here we show some phenotypic and functional characteristics of the induced Treg (iTreg) cell in cultures with PBL from the patients. We analyzed those Tregs for their suppressive function in separate CTL generation assays. We observed that iTreg cells under different conditions do not uniformly express CD25, FoxP3, PDL-1 or CTLA 4 as the known surface markers. When analyzed for their functionality, with adjusted number of cells, in suppressing the anti tumor CTL response, a significant difference was observed. The most effective Tregs cells were found to be those isolated from autologous tumor +PBL IVC or from Mart-1 peptide pulsed DC + PBL IVC. Those cells completely blocked the CTL induction and secreted huge amount of IL-10 upon re-stimulation. Further analysis with these different types of iTreg cells in terms of various gene expressions and corresponding protein secretion will be useful to find a target molecule to block such expansions of iTregs or pTregs cells for better therapeutic outcome.