The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-alpha1beta2 (LTalpha1beta2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTalpha1beta2 expression. At this time, CD45+CD4+CD3- cells are the predominant LTalpha1beta2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTalpha1beta2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTalpha1beta2, a role for CD45+CD4+CD3- cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.