Self-tolerance in T cells is a vital self-defense strategy for mammals to specifically respond to invading pathogens. During T cell development in the thymus, thymocytes migrate from the cortex to the medulla to sequentially acquire non-self-reactivity and self-tolerance. This cortex-to-medulla migration is regulated by CCR7-mediated chemokine signaling. Previous studies have identified CCL21 but not CCL19 as a functional ligand for this CCR7-dependent migration. CCL21 in the mouse is encoded by multiple genes, including CCL21Ser-encoding Ccl21a and several CCL21Leu-encoding genes, including Ccl21b. The importance of Ccl21a in thymocyte migration has been demonstrated, whereas the role of CCL21Leu-encoding genes remains unclear. By producing mice specifically deficient in Ccl21b, we show that Ccl21b plays little to no role in the cortex-to-medulla migration of developing thymocytes. CCL21Leu-encoding gene transcripts remain detectable even in the absence of Ccl21b, suggesting that Ccl21b is not a major source of CCL21Leu. We further show that the copy number of CCL21Leu-encoding genes is smaller than the currently estimated copy number in a public database. These findings underscore the predominant role of Ccl21a over Ccl21b in the mouse thymus.