As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.