The objective was to determine if MAC-T phenotype would impact inflammatory signaling and inflammatory gene expression. MAC-T cells were cultured under basal (DMEM 10% fetal bovine serum and 10 µg/mL of insulin) or lactogenic conditions (basal media + 1 μg/mL ovine prolactin, 0.5 μg/mL hydrocortisone, and 10 mM sodium acetate) and mitogen-activated protein kinase (MAPK; ERK, JNK, and p38) phosphorylation and pro-inflammatory gene expression examined in response to tumor necrosis factor α (TNFα). Statistical analysis was assessed via ANOVA and Tukey’s post-hoc analysis at P ≤ 0.05. MAC-T cells were co-stimulated with increasing concentrations of TNFα (0, 10, 30, 100, 300, 1000 pM). Cell lysates were harvested 15 min post-TNFα stimulation and assessed for MAPK phosphorylation via immunoblotting. JNK and p38 phosphorylation increased in a dose-dependent manner; yet the magnitude of JNK and p38 signaling was greater under basal compared with lactogenic conditions. Cells were next stimulated in parallel with TNFα (300 pM) and lysates harvested over time (0, 15, 30, 100, 120, 180 min). JNK and p38 phosphorylation were robust and transient in MAC-T cells stimulated with TNFα over time. Similar to dose-response experiments, JNK and p38 signaling were significantly more robust in MAC-T cells under basal conditions. We next examined inflammatory gene expression in MAC-T cells cultured under basal or lactogenic conditions and co-stimulated in the presence or absence of TNFα (300 pM). RNA was isolated and PCR array performed to evaluate the expression of 83 inflammatory genes. Pro-inflammatory gene expression was increased in MAC-T cells in response to TNFα. Consistent with enhanced MAPK signaling; pro-inflammatory gene expression was significantly increased in MAC-T cells under basal compared with lactogenic conditions. Real-time qPCR was used to validate PCR array findings. Collectively, our data demonstrate that MAC-T cells cultured under basal conditions are more responsive to TNFα. These findings suggest that investigators consider the importance of MAC-T phenotype when designing future inflammation-related studies.