Recent discoveries have identified human tumour tertiary lymphoid structures (TLS) as organ clusters of innate and adaptive immune cells and stromal cells. These structures promote tumour clearance and enhance responses to checkpoint blockade immunotherapy. However, the absence of TLS in conventional mouse tumour models, particularly subcutaneous injection models, limits experimental manipulation and study of TLS development. Here, we describe a mouse subcutaneous tumour model that naturally forms TLS within the tumour-associated-stroma. These TLSs are identified as clusters of B and T cells in close proximity to high endothelial venules. CCL21 is produced in the T cell zone of perivascular TLS, while high expression of CXCL13 is present in the B cell zone. We further identify CXCL9 as key regulator of the tumour microenvironment. CXCL9 forms distinct niches at the tumour periphery and center. We identified immune cells as the main contributor of CXCL9 in tumour microenvironment, while tumour associated stromal cells produce CXCL9 in vasculature. Deficiency in CXCL9 reduces the efficacy of immune checkpoint blockade immunotherapy and TLS formation. Further single-cell RNA-sequencing and adaptive immune receptor sequencing have been performed to address fundamental questions about TLS ontogeny and the role of CXCL9 during TLS maturation, providing insight into mechanisms of their formation and function. This study advances the understanding of underlying mechanisms of TLS formation and maturation and provides essential information on how these structures can be promoted to enhance cancer therapy.