In contrast to peripheral lymph nodes possessing lymphatic and blood vasculature, the spleen in both humans and rodents is largely devoid of functioning lymphatic capillaries. Here it is reported that in mice lacking homeodomain transcription factor Nkx2-3, the spleen contains an extensive network of lymphocyte-filled sacs lined by cells expressing LYVE-1 antigen, a marker associated with lymphatic endothelium cells (LECs). Real-time quantitative PCR analyses of Nkx2-3 mutant spleen revealed a substantial increase of LYVE-1 and podoplanin mRNA levels, without the parallel increase of mRNA for VEGFR-3 (vascular endothelial growth factor receptor Type 3) and Prox1 (Prospero homeobobox protein 1), two markers specific for LECs. Although these structures express VEGFR-2/flk-1, they lack Prox1 protein, indicating their non-LEC endothelial origin. The LYVE-1(+) structures are bordered with ER-TR7(+) fibroblastic reticular cells with small clusters of macrophages expressing MARCO and sialoadhesin. Short-term cell-tracing studies using labeled lymphocytes indicate that these LYVE-1(+) cysts are largely excluded from the systemic circulation. Cells expressing LYVE-1 glycoprotein as putative precursors for such structures are detectable in the spleen of late-stage embryos, and the formation of LYVE-1(+) structures is independent from the activity of lymphotoxin β-receptor. Thus the splenic vascular defects in Nkx2-3 deficiency include the generation of LYVE-1(+) cysts, comprised of endothelial cells without being committed along the LEC lineage.