ABSTRACT Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD), dexamethasone, IL‐10, or rapamycin. However, challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells, with the capacity to dampen T‐helper responses, occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here, we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes, many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover, ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4 + FoxP3 + CD25 + T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively, our data show that monocytes differentiated with IL‐4, GM‐CSF, and ssON generate cells with phenotypic and functional characteristics of tolDCs.