Abstract Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Methodology Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). Results In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. Conclusion FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro. (FBS however (APCs unknown Therefore activation viabilityproliferation viability viability/proliferation cultured plated 2 h 4 LPS µg/mL. µgmL µg/mL . µg mL µg/mL) point TGFβ1, TGFβ1 TGFβ TGF β1, β1 β (TGF)-β1 OPG , (OPG) IL6, IL6 IL 6, 6 (IL)-6 CCL enzymelinked enzyme linked 24h timepoint. timepoint point. time-point twoway two way ANOVA (ANOVA Tukeys Tukey s posthoc post hoc p<0.05. p005 p p<0.05 0 05 (p<0.05) general concentrationdependent dependent fashion contrast TGF-β1 IL-6 (TGF)-β (OPG (IL)- p00 p<0.0 (p<0.05 TGF-β IL- (IL) p0 p<0. (p<0.0 (IL p<0 (p<0. p< (p<0 (p< (p