It is now accepted that gut microbiota are crucial for the development of the mucosal immune system. It has been suggested that differences in early life microbial colonisation may be associated with variable predisposition to allergic, auto immune, and inflammatory diseases. The exact underlying mechanisms are difficult to study in human infants, however, the similarities in physiology and nutritional requirements between pigs and humans suggest that piglets can be good models to elucidate the pathways involved. The aim of this PhD project was to investigate how differences in microbiota, early on in life, could affect immune regulation in telms of regulatory T cells (Tregs), a cell type fundamental for immune homeostasis. Manipulation of gut microbiota was attempted using different housing conditions [specific pathogen-free (SPF) facility and farm], birth environment (indoor and outdoor fmm) as well as with administration of antibiotics and specific microbiota inocula (with simple and complex composition). Furthermore, the effect of nutritional interventions, (inulin, starch and medium-chain triglycerides) with the potential to manipulate gut microbiota, was also studied. The effect of these manipulations on small intestinal Tregs was examined using fluorescent immunohistology. Furthermore, activation-induced Foxp3 expressions on gut and blood CD4 T cells was also investigated using flow cytometry. It was observed that indoor-born piglets were more susceptible to a reduction in Tregs when transfened to an SPF facility than outdoor bom piglets, but treatment with antibiotics reduced gut Tregs of outdoor-bom piglets at the level of those born indoor. On the other hand, colonisation of new-bom piglets with a complex microbiota inoculum reduced gut Tregs in comparison to the simple microbiota inoculum. However, none of the nutritional interventions had a significant effect on Tregs. Furthermore, no activation induced expression ofFoxp3 was observed in either gut or blood CD4 T cells of 5-month old piglets. The results of this Thesis suggest that both environment and direct manipulation of gut microbiota can affect levels of small intestinal Tregs, whereas the effect of nutrition is less clear. A more detailed analysis of small-intestinal microbiota is necessary to confirm that these observations are a result of differences in the microbiome between the groups or whether other possible factors are also involved.