Lymph nodes (LNs) are strategically positioned at dedicated sites throughout the body to facilitate rapid and efficient immunity. Central to the structural integrity and framework of LNs, and the recruitment and positioning of leukocytes therein, are mesenchymal and endothelial lymph node stromal cells (LNSCs). Advances in the last decade have expanded our understanding and appreciation of LNSC heterogeneity, and the role they play in coordinating immunity has grown rapidly. In this review, we will highlight the functional contributions of distinct stromal cell populations during LN development in maintaining immune homeostasis and tolerance and in the activation and control of immune responses.