Human in vitro generated dendritic cells and the exosomes they release are potential tools for the modulation of immune responses. Here, we characterized differently generated monocyte-derived dendritic cells (MDDCs) and their exosomes. Culturing of peripheral CD14+ cells from the same individuals with either interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (conventional MDDCs) or alternatively with IL-4 and IL-3 generated immature MDDCs in 7 days. Fluorescence-activated cell sorting (FACS) analysis showed that the IL-4/IL-3-generated MDDCs had significantly lower percentages of CD1a+, CD40+ and CD80+ cells and a higher percentage of CD86+ cells as compared with conventional MDDCs. In addition, IL-4/IL-3-generated MDDCs had significantly higher densities of major histocompatibility complex (MHC) class I [human leucocyte antigen (HLA)-ABC], MHC class II (HLA-DR), CD11c and the tetraspanin CD81 as compared with conventional MDDCs. In a comparison of their ability to stimulate CD8+ T cells, we found that the IL-4/IL-3 MDDCs were slightly more efficient than the conventional MDDCs at inducing interferon (IFN)-gamma release in response to viral peptides. Exosome morphology was confirmed by electron microscopy and exosome phenotypes were analysed by flow cytometry and western blot. In comparison to exosomes from conventional MDDCs, exosomes from IL-4/IL-3-generated MDDCs showed significantly stronger signals for HLA-ABC, HLA-DR, CD11c, CD63 and CD81. Thus, phenotypically the exosomes largely reflected their MDDCs of origin. When exosomes were loaded with viral peptides, both types of exosomes induced IFN-gamma release from CD8+ T cells. Our findings might have significance for the development of DC- and exosome-based therapies.