CD11b+GR1+ myeloid-derived suppressor cells (MDSC) accumulate in several inflammatory conditions including cancer, infections, or trauma. MDSCs are found in bone marrow and lymphoid organs and suppress both innate and adaptive immune responses. Although mechanisms of suppression are not fully understood, they have been reported to require cell-cell contact and very often implicate L-arginine metabolism. We and others recently observed that lipopolysaccharide (LPS) administration, as other TLR ligands, induces MDSC. In this case, MDSC regulate immune response independently of L-arginine metabolism through heme oxygenase-1 activity. Manipulating MDSC as immunoregulators represents an attractive approach for cancer immunotherapy or transplantation. Herein, we describe methods for expanding and purifying MDSC, as well as in vitro and in vivo techniques to measure their suppressive functions.