The proatherogenic state of obesity is associated with hypertrophied adipocytes that may arise because of deficient adipogenesis. Macrophages infiltrate adipose tissue as a function of obesity and may release factors that attenuate adipogenesis. Macrophage-conditioned medium inhibits human and 3T3-L1 adipocyte differentiation in culture, but underlying molecular mechanisms have yet to be defined. Exposure of 3T3-L1 cells throughout the 8-day period of differentiation to medium conditioned by THP-1 macrophages (THP-1-MacCM) blocked adipogenesis. Triacylglycerol (TG) accumulation and induction of peroxisome proliferator-activated receptor gamma and fatty acid synthase protein levels were inhibited by 59% (n = 4, P < .001), 29% (n = 4, P < .01), and 47% (n = 4, P < .01), respectively. THP-1-MacCM had no effect when added after the first 2 days of differentiation, indicating that early exposure of its targets must be needed to inhibit 3T3-L1 adipogenesis. Cell enumeration revealed a 44% decrease in clonal expansion compared with standard differentiation (n = 3, P < .01). Addition of THP-1-MacCM to 3T3-L1 preadipocytes increased ERK1/2 phosphorylation by 6.5-fold (n = 3, P < .01). PD98059 (an inhibitor of the ERK1/2 pathway) impaired the negative effect of THP-1-MacCM on TG accumulation, indicated by an inhibition of 25% vs 69% (n = 3, P < .001), without altering fatty acid synthase or peroxisome proliferator-activated receptor gamma levels. Our data implicate ERK1/2 as an important signaling mediator for the inhibitory effect of THP-1-MacCM on TG accumulation during 3T3-L1 adipogenesis.