Although the virulence factors of S. aureus have been studied extensively, how the bacterium grows in the host is an understudied area. More broadly, understanding how bacterial physiology and gene expression are affected by the host tissue environment is of great importance when new therapies are developed. Because certain murine hosts are unable to clear S. aureus from their tissues, the bacteria invade diverse anatomical sites including the skeletal system (bones and joints) and the brain, heart, spleen, and liver, all of which have complex three-dimensional architecture as well as physiological and chemical/nutrient gradients. Exploiting the bacterium’s versatility with respect to tissue colonization, we developed a method for single-cell, spatiotemporal analysis of gene expression in host tissues. Such a method captures valuable information that is lost during the bulk analysis of tissues and cells. Using our method, we recently discovered that S. aureus exhibits spatial regulation of nuc gene expression in kidney abscesses (nuc codes for nuclease; a well-characterized S. aureus virulence factor). Current efforts are focused on understanding the mechanism by which the spatial regulation arises, and the biological significance of such division of labor in S. aureus microcolonies.