Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but could also potentially contribute to tumor progression. As a result, there is significant interest in the clinical manipulation of Tregs. However, the mechanisms governing induced Treg (iTreg) differentiation are not fully understood. In the present study, we phenotypically profiled human iTregs during early stages of in vitro differentiation at single-cell level using multiparametric mass cytometry. A panel of 25 metal-conjugated antibodies specific to a range of markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor Foxp3 and characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was selectively upregulated in the iTreg compartment while negatively regulated by Foxp3. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features, and opens possibilities for studying molecular mechanisms of Treg function.