After the initial discovery of human telomerase deficiency in the X-linked form of the bone marrow failure syndrome dyskeratosis congenita, mutations in genes encoding telomerase subunits have been identified in patients with a wide spectrum of disorders. Structure/function studies of disease-linked variants of human telomerase RNA (hTR) or telomerase reverse transcriptase (TERT) have exploited in vitro reconstitution of the enzyme catalytic core and/or a PCR-amplified activity assay readout that would not reflect alterations of cellular RNP assembly efficiency, telomeric primer recognition, and/or repeat addition processivity. Here we used telomerase reconstitution in vivo and direct telomeric-repeat primer extension activity assays to compare the ribonucleoprotein (RNP) assembly and activity properties of disease-linked subunit variants in holoenzyme context. Analysis of a large panel of hTR variants revealed numerous biochemical mechanisms for telomerase loss of function, including reduced association of hTR with TERT, reduced RNP catalytic activity, or loss in fidelity of telomeric repeat synthesis. An absolute correlation exists between hTR loss of function and hematopoietic deficiency, but there is no readily apparent telomerase deficiency imposed by an hTR variant linked to pulmonary fibrosis. Some disease-linked TERT variants have altered properties of holoenzyme assembly or repeat addition processivity, but other TERT variants linked to either pulmonary fibrosis or hematopoietic deficiency retained normal hTR interaction and RNP catalytic activity. Combined with additional hTR structure/function studies, our results establish a new resolution of insight into hTR structural requirements for hTR-TERT interaction and for the catalytic cycle of human telomerase holoenzyme.