The synthesis of human genomes and other gigabase-scale genomes will require new strategies. Here, we realized key steps in our pipeline for building synthetic human chromosomes. We established: (i) the facile transfer of human chromosomes from human cells to mouse embryonic stem cells (assembly cells), where they are haploid, are nonessential, and may be operated on; (ii) the transfer of these human chromosomes from monochromosomal hybrids back into human cells to generate defined, synthetic aneuploidies; and (iii) the elimination of the corresponding endogenous human chromosomes to regenerate diploid cells containing a transferred chromosome. All steps were performed in nontransformed cells without chromothripsis and generated minimal structural variants, insertions, deletions, or single-nucleotide variants.