A germline copy number duplication of chromosome 14q32, which contains
ATG2B
and
GSKIP
, was identified in families with myeloproliferative neoplasm (MPN). Herein, we show that mice lacking both
Atg2b
and
Gskip
, but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of
ATG2B
and
GSKIP
, the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double knockout fetal livers were significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of
Atg2b
or
Gskip
alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that
Atg2b
and
Gskip
play a synergistic effect in maintaining the pool size of HSCs.