Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers . This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II) and MHC-II subsets with different morphologies. Compared with MHC-II chBMDCs, the MHC-II subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-II chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-II chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-II chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-II subset back into the immature phenotype of the MHC-II subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.