Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER). Our data confirm that optimal hTER activity requires the integrity of four short helices (P2a.1, P2a, P2b, and P3) which create the proposed pseudoknot and that features of both the primary sequence and secondary structure in P2b and P3 contribute to optimal function. At least part of the long-range P1 pairing is also required, despite the lack of a known P1 counterpart in rodent TERs. Among the predicted single-stranded regions, we found that J2b/3, portions of J2a/3, and residues in and around the template make sequence-specific contributions to telomerase function. Additionally, we provide evidence that naturally occurring hTER sequence polymorphisms found in some patients with aplastic anemia can inhibit telomerase activity by disrupting critical structures within the hTER core domain.