Non-hematopoietic mesenchymal stromal cells in secondary lymphoid organs play pivotal roles in tissue organization and immune responses by exhibiting specialized features such as the production of lymphoid homeostatic chemokines. However, the maturational process of stromal cells mediated by lymphotoxin-beta receptor (LTβR) signaling, a key for stromal maturation, remains unclear. Taking advantage of a stromal cell line established from mouse lymph node, which can produce a homeostatic chemokine, CXC chemokine ligand (CXCL) 13, by the engagement of LTβR but not by tumor necrosis factor (TNF) receptor (TNFR), we analyzed the details of intracellular signaling events during the maturational process. The activation of both canonical and non-canonical nuclear factor-κB (NF-κB) pathways was essential for CXCL13 induction; however, an excessive amount of non-canonical RelB–p52 complex was still insufficient for CXCL13 gene expression. Under RelB–p52-over-expressed conditions, TNFα could induce a markedly high amount of CXCL13 production, indicating that the downstream of TNFR contains an additional key component of signaling. We also found that protein kinase C activity plays a critical role in this process in addition to the NF-κB pathways. Taken together, it is suggested that the maturation of lymphoid stromal cells mediated by LTβR is accomplished by the cooperation of multiple signaling cascades.