At birth, the newborn mammal enters a highly microbial world, leading to colonization of the gut by trillions of microorganisms. We previously reported that germ-free (GF) mice have altered brain development in the first days of life, suggesting that the arrival of microbes is essential for normal brain development. In this dissertation I characterized the pioneering microbiota of mice and tested how perturbations contribute to intestinal, immune, and brain development. In Aim 1, I mapped the timing of microbe arrival and community composition in perinatal mice. I found that the mouse colon does not contain measurable bacteria prenatally, and postnatal day (P)3 is the first timepoint where the microbiota could be reliably characterized. In Aim 2, I colonized GF dams with a highly simplified microbial community (the altered Schadler flora, ASF) prior to breeding and compared their offspring to those of unmanipulated GF and control mice at P3 and P23. I observed that ASF newborns have a higher bacterial load in the colon than controls but are colonized by a single bacterial species. For some immune and brain measures, ASF newborns are similar to controls, whereas for others they are GF-like or intermediate. The ASF microbiota grows to just 5 bacterial species by weaning (P23), compared to explosive diversification in controls. Microglia density is normalized in ASF weanlings, but several peripheral measures remain GF-like. Although humans are not born GF or colonized with a defined microbiota, we do routinely alter microbial colonization through antibiotic use. Thus, in Aim 3 I administered one of two antibiotic cocktails (one which is absorbed from the intestine and one which is non-absorbable) peripartum to pregnant mouse dams. Antibiotic treatment decimated the maternal microbiota and led to striking changes in the gut microbiota of offspring that persisted to weaning age. Maternal antibiotic exposure markedly altered neonatal colon gene expression, and caused some effects on peripheral immunity and the brain. Taken together, perturbation of the early life microbiota, including by clinically-relevant antibiotic use, significantly influences intestinal, immune, and brain outcomes. These findings highlight the neonatal period as a critical window during which microbial colonization shapes development.