Cell immunotherapy through dendritic cells (DC) presents a hopeful strategy for the treatment of various tumors. The aim of our study was to find which progenitor cells are most suitable for the preparation of dendritic cells in acute lymphoblastic leukemia (ALL) in pediatric patients, whether blasts from bone marrow or dendritic cells generated from peripheral blood mononuclear cells taken at the time of remission after induction chemotherapy. DC generated from the BM blasts of patients with B-ALL and T-ALL (n = 15) at the time of diagnosis expressed low levels of costimulatory molecules and CD markers typical for mature DC. In contrast, DC cultivated from peripheral mononuclear cells of patients (n = 9) had comparable morphology and expression of costimulatory molecules to DC obtained from healthy individuals, which was even higher after tumor lysate pulsing. Autologous lymphocyte proliferation increased after DC blasts lysate pulsation and further after lymphocyte restimulation, showing evidence of induction of specific cytotoxic lymphocytes. When comparing both cell sources for the preparation of DC in patients with ALL, it appears that peripheral mononuclear cells obtained after chemotherapy are more suitable than bone marrow leukemic blasts due to similar morphology, phenotypic, and functional capacity to monocytes of healthy donors. Despite this, it is necessary to take into account individual variability when preparing DC-based vaccines. The final verification of the efficiency of immunotherapy against residual hematopoietic malignant cells in patients with ALL can only be obtained through a clinical study.