Substantial evidence exists that the interactions between the commensal microbiota and the immune system of intestinal tissues during early life may set the stage for later development of the immune system and susceptibility to inflammatory bowel diseases (IBD). Among the numerous immune cells that are involved in the pathogenesis of IBD, invariant natural killer T (iNKT) cells are increasingly recognized to play an important role. The absence of microbiota during early life but not thereafter leads to iNKT cell accumulation in the colon and incurs later life susceptibility to colitis initiated by environmental triggers.  The mechanisms underlying this accumulation of iNKT cells and how they are regulated by the microbiota exclusively during the “neonatal window of opportunity” remains elusive. Consistent with previous studies, we used parabiotic mice to demonstrate that iNKT cells are tissue resident cells in adult life.  This suggests that the iNKT cell niche is therefore established in early life. Therefore, we used a specific model of transgenic mice that allows for inducible depletion of Csf1r and Lyz2 expressing macrophages and showed that macrophages can regulate iNKT cell numbers in peripheral tissues in a very limited period of time in the colon but not other tissues such as spleen and thymus in a pathway that is CD1d-independent. We show that regulation of iNKT cells by macrophages during this specific window of time during early life can influence later life activation of colonic iNKT cells. Further, early life colonic macrophages are regulated by microbiota and produce signal(s) that are currently under investigation using RNA-Seq that may be responsible for the establishment of iNKT cells in the colon. In summary, we have identified colonic macrophages as regulators of iNKT cell residence specifically during the “neonatal window of opportunity” and describe signals regulated by microbiota that may support this process.