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We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4(+) T-helper (Th) and CD8(+) cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO(+)CD62L(+)) and effector memory (CD45RO(+)CD62L(-)) subsets. We evaluated the phenotype of CD4(+) cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet(+)CD119(+)) and Th17 (RORγt(+)CD161(+)) populations, while having no effect on the Th2 population (GATA3(+)CD193(+)/GATA3(+)CD294(+)cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1β), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4(+)FoxP3(+) cells, and an increase of CD25(+)FoxP3(+) and CD39(+)FoxP3(+) Treg in the CD4(+) population. Induction of Treg cells was corroborated by the increased secretion of TGF-β. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.