Updated project metadata. Here we asked whether infiltration of leukemic blasts initiated a response that could be detected in the interstitial fluid phase of the spleen in a rat model known to mimic human acute myeloid leukemia (AML). By cannulating efferent lymphatic vessels, we were able to monitor the response of the spleen microenvironment during leukemia development. Flow cytometric analysis of lymphocytes isolated from spleen lymph showed increased STAT3 and CREB signaling, and proteins related to these pathways were identified with a different profile in leukemic when compared with control spleen lymph. Additionally, SPARC-like 1 protein, recently identified as a promoter of AML cell growth and a biomarker of patient outcome, was locally produced in the spleen and upregulated in the leukemic setting. Thus, interstitial fluid, and its surrogate efferent lymph, can be used to provide unique information about spleen responses and substances released to the general circulation during leukemia development.