The development of genomic instability is an important step toward generating the multiple genetic changes required for cancer. Telomere dysfunction is one of the factors that contribute to tumorigenesis. Telomeres shorten with each cell division in the absence of telomerase. Human mammary epithelial cells (HMECs) obtained from normal human tissue demonstrate two growth phases. After an initial phase of active growth, HMECs exhibit a growth plateau termed selection. However, some cells can emerge from this growth plateau by spontaneously losing expression of the p16INK4a protein. These post-selection HMECs are capable of undergoing an additional 20-50 population doublings in culture. Continued proliferation of these post-selection HMECs leads to further telomere erosion, loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter bridge-fusion- breakage (BFB) cycles, generating massive chromosomal instability before terminating in a population growth plateau termed agonescence. We have found that the chromosome arms carrying the shortest telomeres are those involved in telomere-telomere type rearrangements. In addition, we found that the risk of a particular chromosome being unstable differs between individuals. Most importantly, we identified sister chromatid fusion as a first event in generating genomic instability in HMECs. During post-selection HMEC growth, double strand breaks are generated by both fused chromosomes as well as individual chromosomes with fused chromatids entering BFB cycles. These broken chromosome extremities are able to join other broken ends or eroded telomeres, producing massive chromosomal instability at the later passages of the cell culture. © 2005 Wiley-Liss. Inc.