Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress-or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.Human somatic cells have a limited replicative life span in vitro, known as the Hayflick limit (1). Our laboratory has proposed a two-stage model of cellular senescence. Normal human fibroblasts undergo a limited number of divisions in culture and eventually enter a non-dividing state designated replicative senescence or mortality stage 1 because telomeres have shortened enough to induce a DNA damage signal. In the absence of cell cycle check points (e.g. p53 or p16/Rb), cells can continue to divide until extreme telomere shortening initiates a second block to cell division, mortality stage 2 (2). During mortality stage 2 (crisis), end fusions and chromosome breakage cycles result in cell death. The expression of telomerase at levels sufficient to maintain or elongate telomeres can prevent the telomere shortening responsible for both mortality stages 1 and 2 and results in the immortalization of some cell types such as retinal pigment epithelial cells and foreskin fibroblasts (3, 4). However, other cell types growth-arrest due to inadequate culture conditions (lack of growth factors, micronutrients, or cellcell interactions), termed stasis (stress-or aberrant signalinginduced senescence) (5). This telomere length-independent growth arrest can be bypassed either by adjusting the culture conditions to the needs of the particular cell type or by abrogating the stress-induced check point that triggers growth arrest. WI-38 fetal lung fibroblasts cannot be immortalized by ectopic human telomerase reverse transcriptase (hTERT) 2 under standard conditions but are immortalized in low oxygen with media suppleme...