Non-Hodgkin lymphoma (NHL) is a heterogeneous and highly disseminated disease, but the mechanisms of its growth and dissemination are not well understood. Using a mouse model of this disease, we used multimodal imaging, including intravital microscopy (IVM) combined with bioluminescence, as a powerful tool to better elucidate NHL progression. We injected enhanced green fluorescent protein and luciferase-expressing Eμ-Myc/Arf(-/-) (Cdkn2a(-/-)) mouse lymphoma cells (EL-Arf(-/-)) into C57BL/6NCrl mice intravenously. Long-term observation inside a peripheral lymph node was enabled by a novel lymph node internal window chamber technique that allows chronic, sequential lymph node imaging under in vivo physiologic conditions. Interestingly, during early stages of tumor progression we found that few if any lymphoma cells homed initially to the inguinal lymph node (ILN), despite clear evidence of lymphoma cells in the bone marrow and spleen. Unexpectedly, we detected a reproducible efflux of lymphoma cells from spleen and bone marrow, concomitant with a massive and synchronous influx of lymphoma cells into the ILN, several days after injection. We confirmed a coordinated efflux/influx of tumor cells by injecting EL-Arf(-/-) lymphoma cells directly into the spleen and observing a burst of lymphoma cells, validating that the burst originated in organs remote from the lymph nodes. Our findings argue that in NHL an efflux of tumor cells from one disease site to another, distant site in which they become established occurs in discrete bursts.