We evaluated the presentation of blood-derived protein Ags by APCs in the thymus. Two conventional dendritic cells (cDCs), the CD8α +Sirpa-CD11chi (CD8α+ cDC) and the CD8α-Sirpα+CD11chi (Sirpa + cDC), were previously identified as presenting MHC class II bound peptides from hen egg white lysozyme (HEL) injected intravenously. All thymic APCs acquired the injected HEL, with the plasmacytoid dendritic cell being the best, followed by the Sirpα+ cDC and the CD8α+ cDC. Both cDCs induced to similar extent negative selection and regulatory T cells in HEL TCR transgenic mice, indicating a redundant role of the two cDC subsets in the presentation of blood-borne HEL. Immature dendritic cells or plasmacytoid dendritic cells were considerably less efficient. Batf3 -/- mice, with significantly reduced numbers of CD8α + cDCs, were not impaired in HEL presentation by I-Ak molecules of thymic APCs. Lastly, clodronate liposome treatment of TCR transgenic mice depleted blood APCs including Sirpα+ cDCs without affecting the number of thymic APCs. In such treated mice, there was no effect on negative selection or regulatory T cells in mice when administering HEL, indicating that the T cell responses were mediated primarily by the cDCs localized in the thymus.