CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are crucial for the development and organisation of lymph nodes and gut associated lymphoid tissues. In this report, we characterise their appearance in the developing spleen and highlight their importance in relation to the development of splenic T cell zones. LTi cells were detected in embryonic spleen from embryonic day 13, although their progenitors were present at embryonic day 12. These cells clustered initially around splenic blood vessels in a lymphotoxin (LT)-independent manner, but up-regulation of VCAM-1 expression on stromal cells associated with the blood vessels was LT dependent. After birth, T cell colonisation of these clusters to form nascent white pulp areas was also LT dependent. Transfer experiments reconstituting RAG(-/-) mice with either WT or LTalpha(-/-) splenocytes demonstrated that lymphocyte expression of LT was not essential for the organisation of a discrete CD3(+) T cell zone with localised podoplanin and CCL21 expression. Our studies indicate that a combination of LT signals from LTi cells and LT-independent signals from lymphocytes is sufficient for expression of podoplanin and CCL21 on splenic T cell zone stroma and subsequent T cell organisation.