While human embryonic stem cells (hESCs) may one day facilitate the treatment of degenerative diseases requiring cell replacement therapy, the success of regenerative medicine is predicated on overcoming the rejection of replacement tissues. Given the role played by dendritic cells (DCs) in the establishment of immunological tolerance, we have proposed that DC, rendered tolerogenic during their differentiation from hESC, might predispose recipients to accept replacement tissues. As a first step towards this goal, we demonstrate that DC differentiated from H1 hESCs (H1-DCs) are particularly responsive to the immunosuppressive agent rapamycin compared to monocyte-derived DC (moDC). While rapamycin had only modest impact on the phenotype and function of moDC, H1-DC failed to upregulate CD40 upon maturation and displayed reduced immunostimulatory capacity. Furthermore, coculture of naïve allogeneic T cells with rapamycin-treated H1-DC promoted an increased appearance of CD25(hi) Foxp3+ regulatory T cells, compared to moDC. Our findings suggest that conditioning of hESC-derived DC with rapamycin favours a tolerogenic phenotype.