Tumors may escape from immune control by the induction of CD11b⁺Gr-1⁺ myeloid suppressor cells in the spleen. In this study, we demonstrate that this cell population can be subdivided into a CD11b^hiGr-1^intSSC^loLy6G^negM-CSFR^int immature monocytic fraction and a CD11b^hi+Gr-1^hiSSC^hiLy6G^hiM-CSFR^neg granulocytic fraction. Upon in vitro culture, the monocytic CD11b⁺Gr-1⁺ cell fraction is sufficient for cytotoxic T lymphocyte (CTL) suppression, which is linked to the gradual differentiation of these monocytic cells into mature F4/80⁺ CD68⁺ macrophages. These CTL-suppressive macrophages are alternatively activated (M2), as demonstrated by the expression of known and novel M2 signature genes. In search of M2-associated genes involved in the suppressive activity, it is shown that stimulation of peroxisome proliferator-activated receptor γ (PPARγ) and inhibition of phospholipase A₂ (PLA₂) activity cooperate to alleviate CTL suppression. Of importance, purified tumor-associated macrophages display a similar M2 phenotype and are suppressive for antitumor CTLs, via a mechanism that can be almost completely reversed by PPARγ ligands. Overall, our data identify PLA₂ and especially PPARγ as new potential therapeutic targets to subvert macrophage-mediated CTL suppression in cancer.