During murine embryogenesis, the formation of Peyer's patches (PPs) is initiated by CD45(+)CD4(+)CD3(-) lymphoid tissue inducers that trigger adhesion molecule expression and specific chemokine production from an organizing stromal cell population through ligation of the lymphotoxin-beta receptor. However, the steps involved in the development of lymph nodes (LNs) are less clear than those of PPs, and the characteristics of the organizing cells within the LN anlagen have yet to be documented. In this study, we show for the first time that the early anlage is bordered by an endothelial layer that retains a mixed lymphatic and blood vascular phenotype up to embryonic day 16.5. This in turn encompasses CD45(+)CD4(+)CD3(-) cells interspersed with ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1, lymphotoxin-beta receptor-positive, chemokine-producing cells analogous to the organizing population previously observed in PPs. Moreover, these LN organizers also express the TNF family member, TRANCE. Lastly, we show that the ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1 cells present in peripheral and mesenteric LN form two discrete populations expressing either intermediate or high levels of these adhesion molecules but that the former population is specifically reduced in PLN. These findings provide a possible explanation for the well-known differences in developmental requirements for nodes at peripheral or mesenteric locations.