Abstract Background The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process. Methods Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. Results We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. Conclusions Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.