Abstract The immune system faces unique challenges in the period immediately following birth. Thrust from a sterile fetal environment, it is abruptly exposed to an immense array of foreign antigens, the major burden of which is in the form of the microbiota newly colonizing the gastrointestinal tract. Adaptive immunity at the post-natal stage is largely immature, and the newborn’s immune system relies initially on innate immune effectors for protection against infections. In the early post-natal period, increasing numbers of innate and adaptive lymphocytes mature in the thymus and migrate to populate peripheral lymphoid organs including intestinal mucosal tissue. Intestinal microbiota regulate the maturation of the immune system at mucosal sites and also influence host physiology and function beyond the mucosal tract such as the brain and the pancreas. However, whether and how intestinal microbial colonization influences immune cell development in the thymus remains unknown. Here we show that the thymus of neonatal mice harbor precursor cells with distinct gene expression profiles compared to adult thymocytes that could potentially favor the development of innate-like lymphocytes. There are discrete patterns of distribution of PLZF and RORgt expressing thymic cell subsets and the prevalence of these cells is influenced by microbial colonization status of the neonatal intestine. Finally, we propose that migratory dendritic cells are one mediator of thymic-intestine crosstalk that transmit microbiota-specific signal to influence development of these thymic subsets. Thus, intestinal microbiota regulate yet another fundamental aspect of immune function- the development of lymphocytes itself in the neonatal thymus.