Modulation of the composition of the intestinal microbiota with probiotics could possibly offer a way of prevention or management of allergic diseases. The objective of this study was to determine the immunomodulating effects of various multispecies probiotic combinations in vitro, as preamble to application in vivo. Multispecies probiotic combinations were formulated and tested for their effects on in vitro cytokine production by human mononuclear cells and were compared to products that already have shown beneficial effects in vivo. All 4 tested combinations of probiotics showed a 40-71% decrease of Th2 cytokine production (IL-4, IL-5, and IL-13) and a variable increase of Th1 (IFN-γ) and Treg cytokine (IL-10) production compared to the medium. A specific probiotic mixture that contained Bifidobacterium breve W25, Bifidobacterium lactis ATCC SD 5219, B. lactis ATCC SD 5220, Lactobacillus plantarum W62, Lactobacillus salivarius W57 and Lactococcus lactis W19 was superior in its stimulating effect on IL-10 production (significant better than the other tested combinations; P=0.001). Modulation of in vitro cytokine production profiles can be used to differentiate between selected probiotic formulations for their immunomodulatory properties. In the future it should be demonstrated whether the immunomodulatory capacities from the multispecies probiotic formulation with the desired profile will be effective in vivo (in adolescents, followed by application in children).