Tumor necrosis factor alpha (TNFalpha), a pro-inflammatory factor, plays an important role in many inflammatory diseases. Inhibition of p38 is being pursued as a pharmaceutical treatment to reduce TNFalpha release. Since a variety of cytokines and factors may exist at different amounts in patients, we explored how differences in the cytokine environment impact p38 inhibitor potency. Cytokine co-stimulation with LPS was compared against LPS stimulation alone. In both differentiated U937 cells and peripheral monocytes, GM-CSF co-stimulation with LPS increased TNFalpha release and led to an increased residual TNFalpha levels with p38 inhibitor. Adding MEK inhibitor in the presence of p38 inhibitor further reduced TNFalpha release suggesting that the ERK pathway plays a role in GM-CSF induced reduction of the p38 inhibitor potency. When cells were stimulated with different concentrations of LPS and GM-CSF, the minimal TNFalpha level obtained by MEK inhibitor was not dependent on the stimulation condition; while it was dependent on GM-CSF level for p38 inhibitor. TNFalpha release in the presence of combinations of p38 and MEK inhibitors under different stimulation conditions was measured. A linear model was created using the initial relative ERK and p38 phosphorylation levels and p38 and MEK inhibitor concentrations to accurately predict released TNFalpha level, suggesting these four parameters are sufficient to predict TNFalpha levels. We then used the model to show that with same TNFalpha levels, higher ERK pathway activity reduces p38 inhibitor potency. These results suggest that p38 inhibitor will be a more potent anti-TNFalpha therapy for patients with low ERK pathway activity.