Myeloid-derived suppressor cells (MDSCs) have an important role in controlling inflammation. As such, they are both a therapeutic target and, based on the administration of ex vivo-generated MDSCs, a therapeutic tool. However, there are relatively few reports describing methods to generate human MDSCs, and most of them rely on cells obtained from peripheral blood monocytes. We investigated alternative approaches to the generation of MDSCs from hematopoietic progenitors and monocytes. Purified CD34+ hematopoietic progenitors from apheresis products and CD14+ cells isolated from buffy coats were cultured in the presence of different combinations of cytokines. The resulting myeloid cell populations were then characterized phenotypically and functionally. Progenitor cells cultured in the presence of SCF+TPO+FLT3-L+GM-CSF+IL-6 gave rise to both monocytic (M)- and granulocytic (G)-MDSCs but production of the latter was partially inhibited by IL-3. M-MDSCs but not G-MDSCs were obtained by culturing peripheral blood monocytes with GM-CSF+IL-6 or GM-CSF+TGF-β1 for 6 days. CD14 expression was downregulated in the cultured cells. PD-L1 expression at baseline was lower in hematopoietic progenitor cell-derived than in monocyte-derived MDSCs, but was markedly increased in response to stimulation with LPS+IFN-γ. The functionality of the two MDSC subtypes was confirmed in studies of the suppression of allogeneic and mitogen-induced proliferation and by cytokine profiling. Here we describe both the culture conditions that allow the generation of MDSCs and the phenotypical and functional characterization of these cell populations.