Findings regarding cytokine levels in neonates and their impact on the development of allergic diseases are controversial. This might be attributed to different laboratory approaches. To compare cytokine levels in supernatants of whole blood (WB) and mononuclear cell (MC) cultures in response to unspecific and allergen specific stimuli. A total population of n = 25 healthy full-term neonates and n = 25 adults was recruited. WB was diluted 1 in 5 and incubated with phytohaemagglutinine (PHA; 20 μg/ml) and the cows’ milk protein betalactoglobulin (BLG) for 24 and 120 h. In parallel, cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) were isolated, and cells were cultured with PHA and BLG in the same concentrations in a medium supplemented with fetal calf serum (FCS) and in a serum- free medium (only PBMC from adults). The cytokines interferon-γ (IFN-γ), interleukin-10 (IL-10), and IL-13 in the cell culture supernatants were measured using the ELISA technique. IFN-γ and IL-10 levels in response to PHA in supernatants of MC of neonates were significantly lower compared to that in adults (p < 0.05, Wilcoxon two-sample test). IL-13 levels were significantly higher in response to PHA in neonates. In adults, only levels of IL-10 were significantly correlated in WB and PBMC cultures (PHA: rS 0.6; p = 0.002; BLG: rS 0.54; p = 0.005). In neonates, IL-10 (PHA: rS 0.77; p < 0.001; BLG: rS 0.63; p < 0.001) and IFN-γ (PHA: rS 0.48; p = 0.02; BLG: rS 0.4; p < 0.047) were significantly correlated. Supernatants of PBMC cultured with an FCS-supplemented medium showed a significant lower IFN-γ release (PHA 1297 pg/ml; BLG 2762 pg/ml) compared to serum-free cell cultures (PHA 6592.5 pg/ml, p < 0.0001; BLG 14228 pg/ml, p = 0.04). IFN-γ and IL-13 levels in WB and MC supernatants revealed significant differences. Our data indicate the need for thoroughly defined and standardized culture conditions for the detection of in vitro cytokine levels.